( A ) Lipidomic data show that relative content of total C20 : 3 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . increase is greatest in mutants with fat - 4 mutation . ( B ) Lipidomic data show that relative content of total C20 : 4 is increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . increase is greatest in mutants with fat - 4 mutation . ( C ) Lipidomic data show that relative content of total C20 : 5 is slightly increased in atgl - 1 ( xd314 ) ; mec - 4 ( d ) but is dramatically decreased in fat - 4 ( wa14 ) ; mec - 4 ( d ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) , which matches role of FAT - 4 in synthesizing C20 : 5 ( EPA ) . Data information data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± . Number of experiments n = 5 , with about 10 , 000 per analyzed in each experiment .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Lipidomic data show that the relative content of total C20:3 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (B) Lipidomic data show that the relative content of total C20:4 is increased in atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The increase is greatest in mutants with the fat-4 mutation. (C) Lipidomic data show that the relative content of total C20:5 is slightly increased in atgl-1(xd314); mec-4(d) but is dramatically decreased in fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d), which matches the role of FAT-4 in synthesizing C20:5 (EPA). Data information The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


( D ) of C20 : 3 - , C20 : 4 - or C20 : 5 - containing TAGs to total lipids containing same PUFA in different genetic backgrounds . of C20 : 4 - containing TAGs to total lipids containing C20 : 4 is significantly increased in atgl - 1 fat - 4 single mutants . is further increased in atgl - 1 fat - 4 double mutant . Data information : FA : free fatty ; TAG : triacylglycerol ; PL : phospholipids . data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± . Number of experiments n = 5 , with about 10 , 000 per analyzed in each experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) The percentage of C20:3-, C20:4- or C20:5-containing TAGs to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing TAGs to total lipids containing C20:4 is significantly increased in atgl-1 and fat-4 single mutants. The percentage is further increased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


( E ) of C20 : 3 - , C20 : 4 - or C20 : 5 - containing phospholipids to total lipids containing same PUFA in different genetic backgrounds . of C20 : 4 - containing phospholipids to total lipids containing C20 : 4 is significantly decreased in atgl - 1 fat - 4 single mutants . is further decreased in atgl - 1 fat - 4 double mutant . Data information : FA : free fatty ; TAG : triacylglycerol ; PL : phospholipids . data were analyzed using two - way ANOVA with Dunnett ' s multiple comparison . Asterisks denote significant differences as compared to mec - 4 ( d ) . ns : not statistically significant . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± . Number of experiments n = 5 , with about 10 , 000 per analyzed in each experiment .

(E) The percentage of C20:3-, C20:4- or C20:5-containing phospholipids to total lipids containing the same PUFA in different genetic backgrounds. The percentage of C20:4-containing phospholipids to total lipids containing C20:4 is significantly decreased in atgl-1 and fat-4 single mutants. The percentage is further decreased in atgl-1 and fat-4 double mutant. Data information: FA: free fatty acids; TAG: triacylglycerol; PL: phospholipids. The data were analyzed using two-way ANOVA with Dunnett's multiple comparison test. Asterisks denote significant differences as compared to mec-4(d). ns: not statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


( F ) of worms that have three or more surviving touch neurons in different genetic backgrounds . mboa - 7 mutation significantly increases of three or more surviving touch neurons in mec - 4 ( d ) , atgl - 1 ( xd314 ) ; mec - 4 ( d ) , fat - 4 ( wa14 ) ; mec - 4 ( d ) atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . data were analyzed using one - way ANOVA with Bonferroni ' s multiple comparison . Asterisks signify significant differences between groups under crossbars . * * P < 0 . 01 , * * * * P < 0 . 0001 . Data show mean ± . Number of experiments n ≥ 4 , with at least 50 per analyzed in each experiment .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The percentage of worms that have three or more surviving touch neurons in different genetic backgrounds. mboa-7 mutation significantly increases the percentage of three or more surviving touch neurons in mec-4(d), atgl-1(xd314); mec-4(d), fat-4(wa14); mec-4(d) and atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Bonferroni's multiple comparison test. Asterisks signify significant differences between the groups under the crossbars. **P < 0.01, ****P < 0.0001. Data show mean ± SEM. Number of experiments n ≥ 4, with at least 50 animals per strain analyzed in each experiment.


( G ) of C20 : 4 - containing phospholipids to total lipids that have C20 : 4 is dramatically decreased in mboa - 7 ( gk399 ) ; atgl - 1 ( xd314 ) ; fat - 4 ( wa14 ) ; mec - 4 ( d ) . data were analyzed using one - way ANOVA with Dunnett ' s multiple comparison . Asterisks signify significant differences as compared to mec - 4 ( d ) . * * * P < 0 . 001 , * * * * P < 0 . 0001 . Data show mean ± . Number of experiments n = 5 , with about 10 , 000 per analyzed in each experiment .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) The percentage of C20:4-containing phospholipids to total lipids that have C20:4 is dramatically decreased in mboa-7(gk399); atgl-1(xd314); fat-4(wa14); mec-4(d). The data were analyzed using one-way ANOVA with Dunnett's multiple comparison test. Asterisks signify significant differences as compared to mec-4(d). ***P < 0.001, ****P < 0.0001. Data show mean ± SEM. Number of experiments n=5, with about 10,000 animals per strain analyzed in each experiment.


B . of PNRC1 expression in normal ( blue circles ) cancer ( red circles ) samples by Real Time PCR .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

B. Quantification of PNRC1 expression levels in primary normal (blue circles) and cancer (red circles) samples by Real Time PCR.


Immunofluorescence stainings of PNRC1 ( red ) Ki - 67 ( green ) performed on a healthy colon ( C ) DAPI was used to stain nuclei . Scale bars : 50µm .

O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a primary healthy colon (C) DAPI was used to stain cell nuclei. Scale bars:50µm.


Immunofluorescence stainings of PNRC1 ( red ) Ki - 67 ( green ) performed on a malignant lymphoma ( D ) . DAPI was used to stain nuclei . Scale bars : 50µm .

O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescence stainings of PNRC1 (red) and Ki-67 (green) performed on a malignant lymphoma (D). DAPI was used to stain cell nuclei. Scale bars:50µm.


E . Proliferation curves of HeLa transfected with HRASG12V ( R ) , PNRC1 ( P ) , their combination ( RP ) or a LacZ control ( L ) . results shown are average ± SD of 2 biological replicates with 3 technical replicates each . A one - way ANOVA with Tukey multiple comparison was performed for 72h dataset ; statistically significant comparisons are as follows : L vs R ( p < 0 . 002 ) , L vs P ( p < 0 . 02 ) , R vs P ( p < 0 . 0002 ) R vs RP ( p < 0 . 0004 )

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Proliferation curves of HeLa cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each. A one-way ANOVA with Tukey multiple comparison test was performed for 72h dataset; the statistically significant comparisons are as follows: L vs R (p<0.002), L vs P (p<0.02), R vs P (p<0.0002) and R vs RP (p<0.0004)


F . Proliferation curves of MCF7 transfected with HRASG12V ( R ) , PNRC1 ( P ) , their combination ( RP ) or a LacZ control ( L ) . results shown are average ± SD of 2 biological replicates with 3 technical replicates each .

O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Proliferation curves of MCF7 cells transfected with HRASG12V (R), PNRC1 (P), their combination (RP) or a LacZ control (L). The results shown are the average ± SD of 2 biological replicates with 3 technical replicates each.


G . Proliferation curves of KRASG12S - mutated A549 transfected with PNRC1 or LacZ control . A representative experiment with average ± SD of 3 technical replicates is shown . An unpaired t - was performed for 144h dataset ( p < 0 . 0003 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Proliferation curves of KRASG12S-mutated A549 cells transfected with PNRC1 or LacZ control. A representative experiment with the average ± SD of 3 technical replicates is shown. An unpaired t-test was performed for 144h dataset (p<0.0003).


H . Proliferation curves of BJ - T transduced with a non - targeting or a PNRC1 - specific shRNA construct . Results are shown as average ± SD of 3 biological replicates with 3 technical replicates each . An unpaired t - was performed for each time point ( * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Proliferation curves of BJ-T cells transduced with a non-targeting or a PNRC1-specific shRNA construct. Results are shown as the average ± SD of 3 biological replicates with 3 technical replicates each. An unpaired t-test was performed for each time point (** p<0.01, *** p<0.005, **** p<0.001).


A . Click - iT imaging performed on HeLa expressing RFP - PNRC1 or RFP control . Confocal of transfected pulsed with EU for 16 hours were collected at indicated time points after EU removal ( green : Alexa 488 - EU , red : RFP , blue : DAPI , scale bar : 5µm ) . RFP - PNRC1 - transfected are indicated with arrowheads .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

A. Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1 or RFP control. Confocal images of transfected cells pulsed with EU for 16 hours were collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1-transfected cells are indicated with arrowheads.


Confocal microscopy of HeLa expressing GFP - PNRC1 stained with antibodies against NPM1 ( B ) nucleolar ( nuclei are stained in blue with DAPI , scale bar : 5µm ) .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against NPM1 (B) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm).


Confocal microscopy of HeLa expressing GFP - PNRC1 stained with antibodies against UBF1 ( C ) nucleolar ( nuclei are stained in blue with DAPI , scale bar : 5µm ) . A magnification of merged channel is provided for UBF1 .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O

Confocal microscopy images of HeLa cells expressing GFP-PNRC1 and stained with antibodies against UBF1 (C) nucleolar proteins (nuclei are stained in blue with DAPI, scale bar: 5µm). A magnification of the merged channel images is provided for UBF1 staining.


D . Agarose / formaldehyde gel separation of neotranscribed rRNAs collected from LacZ PNRC1 - expressing pulsed for 15 minutes with 3H - uridine chased for reported time points ( minutes ) . Samples were loaded according to amount of incorporated 3H , quantified by liquid scintillation . FU was included as a control of a blocked rRNA processing .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Agarose/formaldehyde gel separation of neotranscribed rRNAs collected from LacZ and PNRC1-expressing cells pulsed for 15 minutes with 3H-uridine and chased for the reported time points (minutes). Samples were loaded according to the amount of incorporated 3H, quantified by liquid scintillation. FU was included as a control of a blocked rRNA processing.


E . Ratios between of 47S pre - rRNA band bands belonging to 32S or 28S rRNA species relative to panel ( D ) measured at 60 minutes after chase for both LacZ or PNRC1 - expressing .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O

E. Ratios between the intensity of 47S pre-rRNA band and bands belonging to 32S or 28S rRNA species relative to panel (D) and measured at 60 minutes after the chase for both LacZ or PNRC1-expressing cells.


F . Real Time PCR of 5 ' - ETS steady - state in HeLa expressing PNRC1 or GFP control . average ± SD of 3 biological replicates is shown .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

F. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1 or GFP control. The average ± SD of 3 biological replicates is shown.


A - B . Coomassie blue western blot of SDS - PAGE gel corresponding to PNRC1 IP . Red arrows represent gel areas excised both from IgG IP lanes for mass spectrometry . Input , unbound IP fractions were loaded as reported . band corresponding to PNRC1 is indicated .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

A-B. Coomassie blue staining and western blot analysis of the SDS-PAGE gel corresponding to PNRC1 IP. Red arrows represent the gel areas excised both from the IgG and IP lanes for mass spectrometry analysis. Input, unbound and IP fractions were loaded as reported. The protein band corresponding to PNRC1 is indicated.


C . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1 with a specific anti - HA antibody in absence or presence of 1mg / ml RNaseA .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody in absence or presence of 1mg/ml RNaseA.


D . Co - immunoprecipitation of DCP1α - interacting performed on HA - PNRC1 - expressing HeLa with a specific anti - DCP1α antibody .

O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O

D. Co-immunoprecipitation of DCP1α-interacting proteins performed on HA-PNRC1-expressing HeLa cells with a specific anti-DCP1α antibody.


E . Co - immunoprecipitation performed on wild - type HeLa with an anti - PNRC1 antibody probed for DCP1α .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O

E. Co-immunoprecipitation performed on wild-type HeLa cells with an anti-PNRC1 antibody and probed for DCP1α.


F - G . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1 with a specific anti - HA antibody probed with antibodies specific for involved in decapping . arrow indicates specific DCP2 band , as reported in datasheet of antibody employed .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

F-G. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1 with a specific anti-HA antibody and probed with antibodies specific for proteins involved in RNA decapping. The white arrow indicates the specific DCP2 band, as reported in the datasheet of the antibody employed.


A . Confocal microscopy of RFP or RFP - PNRC1 - expressing stained for DCP1α , DDX6 LSM1 . RFP - PNRC1 - transfected are indicated with arrowheads . Scale bar : 10µm . B . of classified according to P - localization of three stained . average ± SD of 3 independent experiments is reported .

O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Confocal microscopy images of RFP or RFP-PNRC1-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1-transfected cells are indicated with arrowheads. Scale bar: 10µm. B. Quantification of cells classified according to the P-body localization of the three stained proteins. The average ± SD of 3 independent experiments is reported.


C . Nucleolar fractionation performed on LacZ or PNRC1 - expressing HeLa . Total lysates , ( Cyto ) , nucleoplasmic ( Nucl ) nucleolar ( No ) fractions were collected . UBF1 , PolII MEK2 were used as markers for nucleolar , non - nucleolar fractions , respectively . electrophoretic mobility of in nucleoplasmic lanes is slightly diminished due to lysis buffer composition . D . Densitometric of DCP1α nucleolar pool obtained from western blot shown in panel ( C ) normalized upon amount of nucleolar UBF1 of DCP1α in input fraction .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O

C. Nucleolar fractionation performed on LacZ or PNRC1-expressing HeLa cells. Total lysates, cytoplasmic (Cyto), nucleoplasmic (Nucl) and nucleolar (No) fractions were collected. UBF1, RNA PolII and MEK2 were used as markers for the nucleolar, non-nucleolar and cytoplasmic fractions, respectively. The electrophoretic mobility of proteins in the nucleoplasmic lanes is slightly diminished due to nuclear lysis buffer composition. D. Densitometric quantification of DCP1α nucleolar pool obtained from the western blot shown in panel (C) and normalized upon the amount of nucleolar UBF1 and the levels of DCP1α in the input fraction.


E . Confocal microscopy of HeLa silenced for endogenous DCP1α expressing a shRNA - resistant GFP - DCP1α in combination with RFP or RFP - PNRC1 transgenes ( green : GFP , red : RFP , blue : DAPI , scale bar : 5µm ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

E. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing a shRNA-resistant GFP-DCP1α in combination with RFP or RFP-PNRC1 transgenes (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).


A . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1WT , HA - PNRC1W300A or HA - PNRC1ΔNLS with a specific anti - HA antibody probed with anti - NPM1 anti DCP1α antibodies .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O

A. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS with a specific anti-HA antibody and probed with anti-NPM1 and anti DCP1α antibodies.


B - C . Co - immunoprecipitation performed on HeLa expressing HA - PNRC1W300A mutant with a specific anti - HA antibody probed with antibodies specific for PNRC1 - interacting . arrow indicates specific DCP2 band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O

B-C. Co-immunoprecipitation performed on HeLa cells expressing HA-PNRC1W300A mutant with a specific anti-HA antibody and probed with antibodies specific for PNRC1-interacting proteins. The white arrow indicates the specific DCP2 band.


D . Confocal microscopy of HeLa silenced for endogenous DCP1α expressing an shRNA - resistant GFP - DCP1α in combination with RFP , RFP - PNRC1WT or RFP - PNRC1W300A mutant ( green : GFP , red : RFP , blue : DAPI , scale bar : 5µm ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

D. Confocal microscopy images of HeLa cells silenced for the endogenous DCP1α and expressing an shRNA-resistant GFP-DCP1α in combination with RFP, RFP-PNRC1WT or RFP-PNRC1W300A mutant (green: GFP, red: RFP, blue: DAPI, scale bar: 5µm).


E . Confocal microscopy of RFP , RFP - PNRC1 or RFP - PNRC1W300A - expressing stained for DCP1α , DDX6 LSM1 . RFP - PNRC1WT or RFP - PNRC1W300A - transfected are indicated with arrowheads . Scale bar :

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O

E. Confocal microscopy images of RFP, RFP-PNRC1 or RFP-PNRC1W300A-expressing cells stained for DCP1α, DDX6 and LSM1. RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads. Scale bar: 10µm


F . of HeLa expressing RFP - PNRC1W300A classified according to P - localization of three stained . average of 3 independent experiments ± SD is reported .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Quantification of HeLa cells expressing RFP-PNRC1W300A classified according to the P-body localization of the three stained proteins. The average of 3 independent experiments ± SD is reported.


G . Co - immunoprecipitation performed on HeLa expressing LacZ control , HA - PNRC1WT , HA - PNRC1W300A or HA - PNRC1ΔNLS constructs with a specific anti - DCP1α antibody probed with anti - PNRC1 anti - NPM1 antibodies .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O

G. Co-immunoprecipitation performed on HeLa cells expressing LacZ control, HA-PNRC1WT, HA-PNRC1W300A or HA-PNRC1ΔNLS constructs with a specific anti-DCP1α antibody and probed with anti-PNRC1 and anti-NPM1 antibodies.


A . Confocal of a Click - iT imaging performed on HeLa expressing RFP - PNRC1W300A pulsed with EU for 16 hours collected at indicated time points after EU removal ( green : Alexa 488 - EU , red : RFP , blue : DAPI , scale bar : 5µm ) . RFP - PNRC1WT or RFP - PNRC1W300A - transfected are indicated with arrowheads .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O

A. Confocal images of a Click-iT RNA imaging assay performed on HeLa cells expressing RFP-PNRC1W300A pulsed with EU for 16 hours and collected at the indicated time points after EU removal (green: Alexa 488-EU, red: RFP, blue: DAPI, scale bar: 5µm). RFP-PNRC1WT or RFP-PNRC1W300A-transfected cells are indicated with arrowheads.


B . Real Time PCR of 5 ' - ETS steady - state in HeLa expressing PNRC1W300A or GFP control . average of 3 independent experiments ± SD is reported .

B. Real Time PCR quantification of 5'-ETS steady-state levels in HeLa cells expressing PNRC1W300A or GFP control. The average of 3 independent experiments ± SD is reported.


C . of 5 ' - ETS in HeLa infected with an anti - DCP2 or a non - targeting shRNA expressing GFP , PNRC1WT or PNRC1W300A transgenes . average of 2 independent experiments ± SD is reported .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O

C. Quantification of 5'-ETS RNA levels in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average of 2 independent experiments ± SD is reported.


of U3 ( D ) snoRNAs immunoprecipitated by anti - m3G cap antibody measured by Real Time PCR . Values represents average of two biological replicates with three technical replicates

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Levels of U3 (D) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR. Values represents the average of two biological replicates with three technical replicates and are


of U8 ( E ) snoRNAs immunoprecipitated by anti - m3G cap antibody measured by Real Time PCR .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

Levels of U8 (E) snoRNAs immunoprecipitated by the anti-m3G cap antibody measured by Real Time PCR.


F . of U3 snoRNA immunoprecipitated by anti - m3G cap antibody in HeLa infected with an anti - DCP2 or a non - targeting shRNA expressing GFP , PNRC1WT or PNRC1W300A transgenes . average ± SD of 2 independent experiments with three technical replicates is reported .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

F. Levels of U3 snoRNA immunoprecipitated by the anti-m3G cap antibody in HeLa cells infected with an anti-DCP2 or a non-targeting shRNA and expressing GFP, PNRC1WT or PNRC1W300A transgenes. The average ± SD of 2 independent experiments with three technical replicates is reported.


G . Western blot of input Flag - IP fractions of RIP experiment . H . of U3 , U8 U1 co - immunoprecipitating with GFP , PNRC1WT or PNRC1W300A . Results are shown as average ± SD of two biological replicates .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

G. Western blot analysis of input and Flag-IP fractions of RIP experiment. H. RNA levels of U3, U8 and U1 co-immunoprecipitating with GFP, PNRC1WT or PNRC1W300A. Results are shown as the average ± SD of two biological replicates.


( A ) Yeast two - hybrid assays to for interaction between Munc18 - 1 ERK1 / 2 . pGAL4 - Munc18 - 1 ( M18 ) pGAL4 - Empty ( control ) were used as baits ERK1 ERK2 as prey . Both ERK1 ERK2 bind Munc18 - 1 allow growth on selection plates ( minus Histidine ( - HIS ) minus Histidine Adenosine ( - ADE ) ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(A) Yeast two-hybrid assays to test for interaction between Munc18-1 and ERK1/2. pGAL4-Munc18-1 (M18) and pGAL4-Empty (control) were used as baits and ERK1 and ERK2 as prey. Both ERK1 and ERK2 bind Munc18-1 and allow growth on selection plates (minus Histidine (-HIS) and minus Histidine and Adenosine (-ADE)).


( B ) In vitro kinase assays showing Munc18 - 1 phosphorylation by ERK . Radioactive phosphoblot ( 32P ) immunoblot ( IB ) of in vitro ERK kinase using immunoprecipitatedMunc18WT , Munc184A ( M18T78A , S158A , S241A , T574A ) , Munc183A ( M18S158A , S241A , T574A ) , Munc18AA ( M18S241A , T574A ) from HEK293T incubated with recombinant ERK2 . phosphoblot shows phosphorylated Munc18 - 1 ( pMunc18 - 1 ) auto - phosphorylated ERK2 ( pERK2 ) . ( C ) of phospho - Munc18 - 1 in wild - type Munc18AA mutant expressed as amount of 32P incorporation in Munc18 - 1 normalized to total Munc18 - 1 . Phosphorylation of Munc18WT was set to 100 % . ( Munc18WT : 100 ± 3 % ; Munc18AA : 24 ± 2 % , n = 3 , * p = 0 . 01 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(B) In vitro kinase assays showing Munc18-1 phosphorylation by ERK. Radioactive phosphoblot (32P) and immunoblot (IB) of in vitro ERK kinase assay using immunoprecipitatedMunc18WT, Munc184A (M18T78A,S158A,S241A,T574A), Munc183A (M18S158A,S241A,T574A), and Munc18AA (M18S241A,T574A) from HEK293T cells incubated with recombinant ERK2. The phosphoblot shows phosphorylated Munc18-1 (pMunc18-1) and auto-phosphorylated ERK2 (pERK2).(C) Quantification of phospho-Munc18-1 intensity in wild-type and Munc18AA mutant expressed as the amount of 32P incorporation in Munc18-1 normalized to total Munc18-1 levels. Phosphorylation of Munc18WT was set to 100%. (Munc18WT: 100±3%; Munc18AA: 24±2%, n=3, * p = 0.01).


( D ) ERK1 in vitro kinase showing ERK - dependent 32P - incorporation in Munc18WT immuno - precipitated from HEK293T . Western Blot stained with Munc18 - 1 antibody shows total precipitated Munc18 - 1 were identical in conditions .

O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O

(D) ERK1 in vitro kinase assay showing ERK-dependent 32P-incorporation in Munc18WT immuno-precipitated from HEK293T cells. Western Blot stained with Munc18-1 antibody shows total precipitated Munc18-1 levels were identical in all conditions.


( A ) Experimental setup of fear conditioning paradigm . Mice placed in a fear - conditioning box received no shock ( NS ) , an immediate shock ( IS ) or delayed shock ( DS ) were decapitated 30 min after returning to home cage . Dorsal ventral hippocampi were subjected to fractionation ( P2 ) . DS IS significantly increased of phosphorylated ERK1 / 2 , a hallmark of ERK activation . ( B ) Activity values across three different training paradigms to ascertain that foot shock was received in IS DS group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Experimental setup of fear conditioning paradigm. Mice placed in a fear- conditioning box received no shock (NS), an immediate shock (IS) or delayed shock (DS) and were decapitated 30 min after returning to the home cage. Dorsal and ventral hippocampi were subjected to cellular fractionation (P2). DS and IS significantly increased the levels of phosphorylated ERK1/2, a hallmark of ERK activation.(B) Activity values across three different training paradigms to ascertain that foot shock was received in the IS and DS group.


( D ) Western Blot of P2 synaptosomal fractions of ventral hippocampi from fear conditioning paradigm stained for pM18S241 , total Munc18 - 1 , pSynapsin , total synapsin , pERK GAPDH as loading control . ( E ) Munc18 - 1 phosphorylation in dorsal hippocampus expressed as pM18S241 over total Munc18 - 1 . No shock was set to 1 ( NS : 1 . 0 ± 0 . 19 , n = 5 ; DS : 4 . 01 ± 0 . 39 , n = 5 ; IS : 2 . 88 ± 0 . 37 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( F ) of Munc18 - 1 phosphorylation in ventral hippocampus expressed as pM18S241 divided by total Munc18 - 1 . ( NS : 1 . 0 ± 0 . 32 , n = 5 ; DS : 4 . 39 ± 0 . 55 , n = 5 ; IS : 3 . 62 ± 0 . 50 , n = 5 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . ( G ) of synapsin phosphorylation in dorsal hippocampus expressed as pSynapsin divided by total synapsin . ( NS : 1 . 0 ± 0 . 31 , n = 5 ; DS : 1 . 88 ± 0 . 43 , n = 5 ; IS : 1 . 18 ± 0 . 34 , n = 5 ; n . s . ) . Inset shows of ERK2 phosphorylation in dorsal hippocampus expressed as pERK2 divided by total ERK2 . ( NS : 1 . 0 ± 0 . 11 , n = 5 ; DS : 3 . 51 ± 0 . 41 , n = 5 ; IS : 3 . 98 ± 0 . 24 , n = 5 ; * * p < 0 . 01 ) . ( H ) of synapsin phosphorylation in ventral hippocampus expressed as pSynapsin divided by total synapsin . ( NS : 1 . 0 ± 0 . 20 , n = 5 ; DS : 3 . 32 ± 0 . 46 , n = 5 ; IS : 3 . 30 ± 0 . 60 , n = 5 ; * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western Blot analysis of P2 synaptosomal fractions of ventral hippocampi from the fear conditioning paradigm stained for pM18S241, total Munc18-1, pSynapsin, total synapsin, pERK and GAPDH as loading control.(E) Quantification Munc18-1 phosphorylation in dorsal hippocampus expressed as ratio pM18S241 over total Munc18-1 levels. No shock was set to 1 (NS: 1.0±0.19, n=5; DS: 4.01±0.39, n=5; IS: 2.88±0.37, n=5; **p<0.01; ***p<0.001).(F) Quantification of Munc18-1 phosphorylation in ventral hippocampus expressed as ratio pM18S241 divided by total Munc18-1 levels. (NS: 1.0±0.32, n=5; DS: 4.39±0.55, n=5; IS: 3.62±0.50, n=5; **p<0.01; ***p<0.001).(G) Quantification of synapsin phosphorylation in dorsal hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.31, n=5; DS: 1.88±0.43, n=5; IS: 1.18±0.34, n=5; n.s.). Inset shows quantification of ERK2 phosphorylation in dorsal hippocampus expressed as ratio pERK2 divided by total ERK2 levels. (NS: 1.0±0.11, n=5; DS: 3.51±0.41, n=5; IS: 3.98±0.24, n=5; **p<0.01).(H) Quantification of synapsin phosphorylation in ventral hippocampus expressed as ratio pSynapsin divided by total synapsin levels. (NS: 1.0±0.20, n=5; DS: 3.32±0.46, n=5; IS: 3.30±0.60, n=5; **p<0.01).


( A ) Western Blot of whole lysate of adult mousebrain slices treated with proteasome inhibitor MG132 ( 10 M ) DMSO , GABAA receptor blocker GABAzine ( GBZ , 10 M ) or voltage - gated sodium channel blocker TTX ( 2 M ) for 4 h . Blots were stained for pMunc18S241 , Munc18 - 1 tubulin as loading control . ( B ) of ( A ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as pM18S241 divided by total Munc18 - 1 . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 4 % ; GBZ : 1 . 73 ± 0 . 12 ; TTX : 0 . 052 ± 0 . 002 , n = 4 , * p < 0 . 05 , * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western Blot of whole cell lysate of adult mousebrain slices treated with the proteasome inhibitor MG132 (10 M) and DMSO, the GABAA receptor blocker GABAzine (GBZ, 10 M) or the voltage-gated sodium channel blocker TTX (2 M) for 4 h. Blots were stained for pMunc18S241, Munc18-1 and tubulin as loading control.(B) Quantification of (A). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.4%; GBZ: 1.73 ± 0.12; TTX: 0.052 ± 0.002, n = 4, * p<0.05, ** p<0.01).


( C ) Western Blot of whole lysate of adult mousebrain slices treated with proteasome inhibitor MG132 ( 10 M ) MEK inhibitor PD98059 ( 10 M ) for 4 h . Proteasome block increases of phosphorylated Munc18 - 1 ERK1 / 2 , which can be prevented by MEK inhibition . Blots were stained for pMunc18S241 , Munc18 , pERK1 / 2 ERK1 / 2 . ( D ) of ( C ) . ERK phosphorylation of Munc18 - 1 in adult mousebrain slices expressed as pM18S241 divided by total Munc18 - 1 . Vehicle ( DMSO ) was set to 1 ( DMSO : 100 ± 0 . 8 % ; MG132 : 375 ± 14 . 2 % ; MG132 + PD98059 : 24 ± 3 % , n = 3 , * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western Blot of whole cell lysate of adult mousebrain slices treated with proteasome inhibitor MG132 (10 M) and MEK inhibitor PD98059 (10 M) for 4 h. Proteasome block increases the levels of phosphorylated Munc18-1 and ERK1/2, which can be prevented by MEK inhibition. Blots were stained for pMunc18S241, Munc18, pERK1/2 and ERK1/2.(D) Quantification of (C). ERK phosphorylation of Munc18-1 in adult mousebrain slices expressed as ratio pM18S241 divided by total Munc18-1 levels. Vehicle (DMSO) was set to 1 (DMSO: 100 ± 0.8%; MG132: 375 ± 14.2%; MG132 + PD98059: 24 ± 3%, n = 3, ** p<0.01).


( E ) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 ( 1 M ) for 5 min or BDNF ( 100ng / ml ) for 48 h in presence of 2 M TTX stained for pMunc18S241 , Munc18 , pERK tubulin as loading control .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

(E) DIV14 rat cortical cultures treated with type 2 mGluR agonist LY379268 (1 M) for 5 min or BDNF (100ng/ml) for 48 h in the presence of 2 M TTX and stained for pMunc18S241, Munc18, pERK and tubulin as loading control.


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( A ) Amplitude of excitatory synaptic response ( EPSC ) ( M18WT : 1 . 37 ± 0 . 21 nA , n = 35 ; M18S241A : 2 . 53 ± 0 . 31 nA , n = 45 ; M18S241D : 0 . 24 ± 0 . 05 nA , n = 17 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(A) Amplitude of excitatory synaptic response (EPSC) (M18WT: 1.37±0.21 nA, n=35; M18S241A: 2.53±0.31 nA, n=45; M18S241D: 0.24±0.05 nA, n=17, ***p<0.001, **p<0.01, *p<0.05).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( B ) of that do not respond to electrical stimulation ( M18WT : 15 . 5 % ; M18S241A : 0 % ; M18S241D : 56 . 9 % ) . These are not included in ( A ) . Insert : representative recording of single EPSCs . Scale bar represents 10 ms 0 . 5 nA .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(B) Percentage of cells that do not respond to electrical stimulation (M18WT: 15.5%; M18S241A: 0%; M18S241D: 56.9%). These cells are not included in (A). Insert: representative recording of single EPSCs. Scale bar represents 10 ms and 0.5 nA.


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( C ) Typical examples of spontaneous vesicle release . Scale bar represents 0 . 1 s 25 pA . ( D ) Spontaneous release frequency ( M18WT : 7 . 05 ± 1 . 28 Hz , n = 32 ; M18S241A : 14 . 26 ± 2 . 04 Hz , n = 37 ; M18S241D : 0 . 64 ± 0 . 29 Hz , n = 11 ; * * * p < 0 . 001 , * * p < 0 . 01 ) . ( E ) Amplitude of spontaneous release events ( M18WT : 15 . 40 ± 1 . 06 pA , n = 17 ; M18S241A : 15 . 03 ± 0 . 61 pA , n = 20 ; M18S241D : 10 . 98 ± 0 . 72 pA , n = 10 ; * * p < 0 . 01 ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(C) Typical examples of spontaneous vesicle release. Scale bar represents 0.1 s and 25 pA.(D) Spontaneous release frequency (M18WT: 7.05 ± 1.28 Hz, n=32; M18S241A: 14.26 ± 2.04 Hz, n=37; M18S241D: 0.64 ± 0.29 Hz, n=11; ***p<0.001, **p<0.01).(E) Amplitude of spontaneous release events (M18WT: 15.40 ± 1.06 pA, n=17; M18S241A: 15.03 ± 0.61 pA, n=20; M18S241D: 10.98 ± 0.72 pA, n=10; **p<0.01).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( F ) of readily releasable pool ( RRP ) assessed with single hyperosmotic sucrose application ( 500 mM , 3 . 5 s , M18WT : 0 . 74 ± 0 . 15 nC , n = 7 ; M18S241A : 1 . 6 ± 0 . 33 nC , n = 10 ; M18S241D : could not be determined ; * p < 0 . 05 ) .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(F) Size of the readily releasable pool (RRP) assessed with single hyperosmotic sucrose application (500 mM, 3.5 s, M18WT: 0.74 ± 0.15 nC, n=7; M18S241A: 1.6 ± 0.33 nC, n=10; M18S241D: could not be determined; * p < 0.05).


Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT , M18S241A or M18S241D . Electrophysiology recordings were performed between DIV14 - 17 . ( G ) Neurons were stimulated with two consecutive pulses with varying inter - stimulus interval to assess release probability . Paired calculated as of second to first synaptic response is plotted as function of inter - stimulus interval ( M18WT , n = 25 ; M18S241A , n = 19 ; M18S241D , n = 17 ) ; * p < 0 . 05 , * * p < 0 . 01 ) . data are expressed as mean ± . See also Figure EV2 EV3 .

O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT, M18S241A or M18S241D. Electrophysiology recordings were performed between DIV14-17.(G) Neurons were stimulated with two consecutive pulses with varying inter-stimulus interval to assess release probability. Paired pulse ratio calculated as ratio of the second to the first synaptic response is plotted as function of inter-stimulus interval (M18WT, n=25; M18S241A, n=19; M18S241D, n=17); * p < 0.05, ** p < 0.01). All data are expressed as mean ± SEM. See also Figure EV2 and EV3.


( A - F ) Munc18 - 1 were measured in glia free cultures of munc18 - 1null mutant neurons expressing M18WT , M18S241A or M18S241D . ( A ) Typical examples showing Munc18 - 1 localization in neurites of M18WT , M18S241A or M18S241Dneurons . MAP2 in blue , VAMP2 in red Munc18 - 1 in green . Scale bar represents 2 m . ( B ) Mean somatic Munc18 - 1 ( M18WT : 1682 ± 206 a . u . , n = 46 ; M18S241A : 1719 ± 238 a . u . , n = 36 ; M18S241D : 582 ± 98 a . u . , n = 33 ; * * * p < 0 . 001 ) . ( C ) Mean synapticMunc18 - 1 ( M18WT : 1281 ± 161 a . u . ; M18S241A : 1390 ± 180 a . u . ; M18S241D : 210 ± 30 a . u . ; * * * p < 0 . 001 ) . ( D ) Mean synapticMunc18 - 1 as a function of radial from soma .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1null mutant neurons expressing M18WT, M18S241A or M18S241D.(A) Typical examples showing Munc18-1 protein localization and intensity in neurites of M18WT, M18S241A or M18S241Dneurons. MAP2 in blue, VAMP2 in red and Munc18-1 in green. Scale bar represents 2 m.(B) Mean somatic Munc18-1 intensity (M18WT: 1682 ± 206 a.u., n = 46; M18S241A: 1719 ± 238 a.u., n = 36; M18S241D: 582 ± 98 a.u., n = 33; *** p < 0.001).(C) Mean synapticMunc18-1 intensity (M18WT: 1281 ± 161 a.u.; M18S241A: 1390 ± 180 a.u.; M18S241D: 210 ± 30 a.u.; *** p < 0.001).(D) Mean synapticMunc18-1 intensity as a function of radial distance from the soma.


( A - F ) Munc18 - 1 were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( E ) Mean synaptic Munc18 - 1 in cultures with 3 times standard number of neurons to increase network activity ( M18WT : 1072 ± 123 a . u . , n = 36 ; M18S241A : 1480 ± 170 a . u . , n = 32 ; M18S241D : 251 ± 70 a . u . , n = 34 ; * p < 0 . 05 , * * * p < 0 . 001 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(E) Mean synaptic Munc18-1 intensity in cultures with 3 times the standard number of neurons to increase network activity (M18WT: 1072 ± 123 a.u., n = 36; M18S241A: 1480 ± 170 a.u., n = 32; M18S241D: 251 ± 70 a.u., n = 34; * p < 0.05, *** p < 0.001).


( A - F ) Munc18 - 1 were measured in glia free cultures of munc18 - 1 null mutant neurons expressing M18WT , M18S241A or M18S241D . ( F ) Mean synaptic Munc18 - 1 in cultures treated with GABAA receptor antagonist Bicuculline ( 40 μM , 48 h ) . ( M18WT : 761 ± 121 a . u . , n = 26 ; M18S241A : 1482 ± 150 a . u . , n = 31 , * * p < 0 . 01 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-F) Munc18-1 protein levels were measured in glia free cultures of munc18-1 null mutant neurons expressing M18WT, M18S241A or M18S241D.(F) Mean synaptic Munc18-1 intensity in cultures treated with GABAA receptor antagonist Bicuculline (40 μM, 48 h). (M18WT: 761 ± 121 a.u., n = 26; M18S241A: 1482 ± 150 a.u., n = 31, ** p < 0.01).


O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Mean synaptic syntaxin intensity in autaptic cultures (M18WT: 875 ± 35, n = 51; M18S241A: 1058 ± 42, n = 46; M18S241D: 458 ± 19, n=40, ** p < 0.01, ***p<0.001).


( H ) Western Blot of lysates from high - density neuronal cultures from munc18 - 1 null mutant mice rescued with M18WT , M18S241A or M18S241D . Prior to lysis , were treated for 6 h with MG132 ( 10 M ) or DMSO as control . Western Blots were stained for Munc18 - 1 tubulin as loading control . ( I ) of ( H ) . Munc18 - 1 were normalized to tubulin . Effect of proteasome inhibition was quantified as between MG132 DMSO treatment of each mutant ( M18WT : 1 . 62 ± 0 . 41 ; M18S241A : 1 . 26 ± 0 . 16 ; M18S241D : 7 . 53 ± 1 . 17 , n = 3 ; * * p < 0 . 01 ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Western Blot of lysates from high-density neuronal cultures from munc18-1 null mutant mice rescued with M18WT, M18S241A or M18S241D. Prior to lysis, cells were treated for 6 h with MG132 (10 M) or DMSO as control. Western Blots were stained for Munc18-1 and tubulin as loading control.(I) Quantification of (H). Munc18-1 levels were normalized to tubulin levels. Effect of proteasome inhibition was quantified as ratio between MG132 and DMSO treatment of each mutant (M18WT: 1.62 ± 0.41; M18S241A: 1.26 ± 0.16; M18S241D: 7.53 ± 1.17, n = 3; **p<0.01).


( J ) Ubiquitination of different Munc18 - 1 mutants in HEK293T . Western Blot of denatured Munc18 - 1 immuno - precipitations from expressing Munc18 - 1 mutants HA - tagged ubiquitin . Prior to lysis , were treated for 8 h with MG132 ( 10 M ) . Ubiquitin covalently attached to Munc18 - 1 can be observed as a smear above characteristic 68 kD Munc18 - 1 band on Western blots stained for HA - tag . ( K ) of immuno - precipitation in ( J ) . Level of Munc18 - 1 ubiquitination was quantified as of immunoprecipitated ubiquitin Munc18 - 1 . M18WT was set to 1 ( M18WT : 1 ± 0 . 06 ; M18S241A : 0 . 84 ± 0 . 57 ; M18S241D : 2 . 70 ± 0 . 07 , n = 3 ; * * p < 0 . 01 ) . data are expressed as mean ± .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Ubiquitination of different Munc18-1 mutants in HEK293T cells. Western Blot of denatured Munc18-1 immuno-precipitations from cells expressing Munc18-1 mutants and HA-tagged ubiquitin. Prior to lysis, cells were treated for 8 h with MG132 (10 M). Ubiquitin covalently attached to Munc18-1 can be observed as a smear above the characteristic 68 kD Munc18-1 band on Western blots stained for HA-tag.(K) Quantification of immuno-precipitation in (J). Level of Munc18-1 ubiquitination was quantified as ratio of immunoprecipitated ubiquitin and Munc18-1 levels. M18WT was set to 1 (M18WT: 1 ± 0.06; M18S241A: 0.84 ± 0.57; M18S241D: 2.70 ± 0.07, n = 3; **p<0.01). All data are expressed as mean ± SEM.


( A ) Western blot from whole lysate of munc18 - 1 null mutant neurons rescued with M18WT incubated with WIN55 , 212 - 2 ( 5 M ) for 30 s . were harvested after indicated time . CB1R activation leads to a rapid transient increase in pERK activity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Western blot from whole cell lysate of munc18-1 null mutant neurons rescued with M18WT and incubated with WIN55,212-2 (5 M) for 30 s. Cells were harvested after the indicated time. CB1R activation leads to a rapid and transient increase in pERK activity.


( B - D ) Hippocampal autaptic neurons of munc18 - 1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A . CB1R agonist WIN55 , 212 - 2 ( 5 M ) was applied at t = 60 s for 30 s . ( B ) Amplitude of excitatory synapse response normalized to t = 0 s . Neurons were stimulated by a single action potential every 30 s for 4 min ( M18WTn = 21 , M18S241An = 24 , * p < 0 . 05 ) . Note broken y - axis . ( C ) of frequency of spontaneous release events after ( t = 180 s ) before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 58 . 0 ± 2 . 3 % , n = 21 ; M18S241A : 84 . 2 ± 5 . 6 % , n = 24 , * p < 0 . 05 ) . ( D ) of amplitude of spontaneous release events after ( t = 240 s ) before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 85 . 0 ± 2 . 7 % , n = 21 ; M18S241A : 83 . 1 ± 2 . 6 % , n = 24 ) .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-D) Hippocampal autaptic neurons of munc18-1null mutant mice were rescued with lentiviruses expressing M18WT or M18S241A. CB1R agonist WIN55,212-2 (5 M) was applied at t = 60 s for 30 s.(B) Amplitude of excitatory synapse response normalized to t=0 s. Neurons were stimulated by a single action potential every 30 s for 4 min (M18WTn = 21, M18S241An = 24, *p<0.05). Note the broken y-axis.(C) Ratio of the frequency of spontaneous release events after (t = 180 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 58.0 ± 2.3 %, n = 21; M18S241A: 84.2 ± 5.6 %, n = 24, * p < 0.05).(D) Ratio of amplitude of spontaneous release events after (t=240 s) and before (t=0 s) WIN55,212-2 application (M18WT: 85.0 ± 2.7 %, n = 21; M18S241A: 83.1 ± 2.6 %, n = 24).


( E - G ) Autaptic neurons of munc18 - 1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55 , 212 - 2 ( 1 M ) , WIN55 , 212 - 2 ( 1 M ) plus AM251 ( 1 M ) or vehicle only ( DMSO ) . ( E ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) or vehicle only ( DMSO ) were applied at t = 60 s for 30 s . ( WT + WIN n = 13 ; WT + DMSO n = 16 , * p < 0 . 05 ) . Note broke Y - axis . ( F ) Paired ( 20 ms interval ) at t = 0 t = 240 s in M18WT neurons superfused with CB1R agonist WIN55 , 212 - 2 ( 1 M ) at t = 60 s for 30 s . WIN55 , 212 - 2 application increases PP ratios indicative of a reduction of synaptic release probability ( t = 0 s : 0 . 94 ± 0 . 1 , n = 13 ; t = 240 s : 1 . 36 ± 0 . 2 , n = 16 , * p < 0 . 05 ) . ( G ) Amplitude of excitatory synapse response normalized to t = 0 s . CB1R agonist WIN55 , 212 - 2 ( 1 M ) was applied together with CB1R antagonist AM251 ( 1 M ) at t = 60 s for 30 s . ( WT + WIN n = 13 , same trace as 6F ; WT + WIN + AM251 n = 15 , * p < 0 . 05 ) . Note broke Y - axis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-G) Autaptic neurons of munc18-1 null mutant mice rescued with lentiviruses expressing M18WT were superfused with WIN55,212-2 (1 M), WIN55,212-2 (1 M) plus AM251 (1 M) or vehicle only (DMSO).(E) Amplitude of excitatory synapse response normalized to t=0 s. CB1R agonist WIN55,212-2 (1 M) or vehicle only (DMSO) were applied at t=60 s for 30 s. (WT + WIN n = 13; WT + DMSO n = 16, * p < 0.05). Note the broke Y-axis.(F) Paired pulse ratio (20 ms interval) at t = 0 and t = 240 s in M18WT neurons superfused with CB1R agonist WIN55,212-2 (1 M) at t = 60 s for 30 s. WIN55,212-2 application increases PP ratios indicative of a reduction of synaptic release probability (t = 0 s: 0.94 ± 0.1, n = 13; t = 240 s: 1.36 ± 0.2, n = 16, * p < 0.05).(G) Amplitude of excitatory synapse response normalized to t = 0 s. CB1R agonist WIN55,212-2 (1 M) was applied together with the CB1R antagonist AM251 (1 M) at t = 60 s for 30 s. (WT + WIN n = 13, same trace as 6F; WT + WIN + AM251 n = 15, * p < 0.05). Note the broke Y-axis.


( H - J ) Hippocampal autaptic neurons of munc18 - 1 null mutant mice were rescued with M18WT or M18S241A . mGluR2 / 3 agonist LY379268 ( 1 M ) was applied by bath - perfusion for 4 min . ( H ) of amplitude of evoked release events after ( t = 240 s ) before ( t = 0 s ) LY379268 application ( M18WT : 55 . 0 ± 4 . 7 % , n = 14 ; M18S241A : 81 . 1 ± 3 . 6 % , n = 16 , * p < 0 . 05 ) . ( I ) of frequency of spontaneous release events after ( t = 240 s ) before ( t = 0 s ) LY379268 application ( M18WT : 63 . 0 ± 5 . 7 % , n = 14 ; M18S241A : 85 . 1 ± 3 . 4 % , n = 16 , * p < 0 . 05 ) . ( J ) of amplitude of spontaneous release events after ( t = 240 s ) before ( t = 0 s ) WIN55 , 212 - 2 application ( M18WT : 81 . 0 ± 1 . 3 % , n = 14 ; M18S241A : 82 . 1 ± 3 . 3 % , n = 16 ) .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-J) Hippocampal autaptic neurons of munc18-1 null mutant mice were rescued with M18WT or M18S241A. mGluR2/3 agonist LY379268 (1 M) was applied by bath-perfusion for 4 min.(H) Ratio of amplitude of evoked release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 55.0 ± 4.7 %, n = 14; M18S241A: 81.1 ± 3.6 %, n = 16, * p < 0.05).(I) Ratio of frequency of spontaneous release events after (t = 240 s) and before (t = 0 s) LY379268 application (M18WT: 63.0 ± 5.7 %, n = 14; M18S241A: 85.1 ± 3.4 %, n = 16, * p < 0.05).(J) Ratio of the amplitude of spontaneous release events after (t = 240 s) and before (t = 0 s) WIN55,212-2 application (M18WT: 81.0 ± 1.3 %, n = 14; M18S241A: 82.1 ± 3.3 %, n = 16).


( A - D ) DSE duration amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre - treatment with PD98059 ( PD ; 10 µM ) compared to 0 . 1 % DMSO ( control ) . ( A ) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between DMSO PD pretreated groups ( DMSO : 196 . 36 + / - 16 . 51 pA , n = 10 ; PD : 196 . 42 + / - 18 . 61 pA , n = 11 , p = 0 . 9 ) . ( B ) DSE in neurons of DMSO pre - treated slices , induced by a 10s depolarization to 0 mV ( arrow ) . Inset : example traces of 2 averaged EPSCs before ( black ) after depolarization ( grey ) . ( C ) DSE in slices pre - treated with PD . Insets : example traces of 2 averaged EPSCs before ( red ) after depolarization ( grey ) . ( D ) Comparison of DSE between control PD . PD pre - treatment limits amplitude duration of DSE . * * = p < 0 . 01 ; unpaired T - with Welch ' s correction . Numbers in bars represent number of neurons in each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) DSE duration and amplitude is decreased in CA1 pyramidal neurons upon ERK pathway inhibition by a pre-treatment with PD98059 (PD; 10 µM) compared to 0.1% DMSO (control).(A) Average EPSC amplitude during 25 s baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 196.36 +/- 16.51 pA, n = 10; PD: 196.42 +/- 18.61 pA, n = 11, p = 0.9).(B) DSE in neurons of DMSO pre-treated slices, induced by a 10s depolarization to 0 mV (arrow). Inset: example traces of 2 averaged EPSCs before (black) and after depolarization (grey).(C) DSE in slices pre-treated with PD. Insets: example traces of 2 averaged EPSCs before (red) and after depolarization (grey).(D) Comparison of DSE between control and PD. PD pre-treatment limits amplitude and duration of DSE. ** = p < 0.01; unpaired T-test with Welch's correction. Numbers in bars represent the number of neurons in each group.


( E - H ) ERK - pathway inhibition by pre - treatment with PD98059 ( PD ; 10 µM ) blocks effect of WIN55 , 212 - 2 ( WIN ; 2 µM ) on amplitude of EPSCs recorded in CA1 pyramidal neurons . ( E ) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between DMSO PD pretreated groups ( DMSO : 295 . 69 + / - 28 . 73 pA , n = 6 ; PD : 270 . 86 + / - 32 . 14 pA , n = 6 , p = 0 . 6 ) . ( F ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of 0 . 1 % DMSO ( control ) pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( black ) last 5 min ( grey ) . ( G ) Effect of WIN application ( grey bar , 30 min ) on EPSC - amplitude in CA1 pyramidal neurons of PD pre - treated slices . Inset : example traces of averaged EPSCs of baseline ( red ) last 5 minutes ( grey ) . ( H ) Comparison of effect of last 5 minutes in both groups shows a significant difference . * = p < 0 . 05 ; unpaired T - . Numbers in bars represent number of slices in each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-H) ERK-pathway inhibition by pre-treatment with PD98059 (PD; 10 µM) blocks the effect of WIN55,212-2 (WIN; 2 µM) on the amplitude of EPSCs recorded in CA1 pyramidal neurons.(E) Average EPSC amplitude during 5 min baseline recording prior to DSE induction shows no significant differences in EPSC amplitude between the DMSO and PD pretreated groups (DMSO: 295.69 +/- 28.73 pA, n = 6; PD: 270.86 +/- 32.14 pA, n = 6, p= 0.6).(F) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of 0.1% DMSO (control) pre-treated slices. Inset: example traces of averaged EPSCs of baseline (black) and the last 5 min (grey).(G) Effect of WIN application (grey bar, 30 min) on EPSC-amplitude in CA1 pyramidal neurons of PD pre-treated slices. Inset: example traces of averaged EPSCs of baseline (red) and the last 5 minutes (grey).(H) Comparison of the effect size of the last 5 minutes in both groups shows a significant difference. * = p < 0.05; unpaired T-test. Numbers in bars represent number of slices in each group.


E ) Caco - 2 were transfected with indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h . Western blotting of lysates is shown .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

E) Caco-2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h. Western blotting of cell lysates is shown.


D ) Graph depicts normalized infection measurements from 2 - 3 independent experiments , each performed in triplicate , from infected Bars represent averages with data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable were transiently transfected with human IFITM plasmids or vector control for 24 h . D ) Graph depicts normalized infection measurements from 2 - 3 independent experiments , each performed in triplicate , from infected Bars represent averages with data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable were transiently transfected with mouse ( m ) IFITM plasmids or vector control for 24 h . C ) Graph depicts normalized infection measurements from 2 - 3 independent experiments , each performed in triplicate , from infected Bars represent averages with data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293T-ACE2-GFP stable cells were transiently transfected with mouse (m) IFITM plasmids or vector control for 24 h. C) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


D ) Graph depicts normalized infection measurements from two experiments with infected as in ( C ) one experiment with MOI 0 . 1 . Bars represent averages with data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in (C) and one experiment with MOI 0.1. Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


U2OS transfected with EGFP indicated IFITM3 constructs or vector control were mixed Calu - 3 transfected with mCherry SARS - CoV - 2 Spike ( S ) or vector control . were co - cultured for 24 h imaged by microscopy to visualize EGFP mCherry as well as nuclei via Hoechst dye . A ) Representative example imaging of indicated co - cultures showing presence of EGFP / mCherry double positive syncytia dependent upon S expression as well as varying effects of IFITM3 constructs . Scale bar indicates 50 μm . B ) of nuclei per EGFP / mCherry double positive syncytia in co - cultures involving indicated IFITM3 constructs as compared to vector control . Bars depict averages of three independent experiments with circles representing data points . For each condition , nuclei within 50 - 75 syncytia were counted . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu-3 cells transfected with mCherry and SARS-CoV-2 Spike (S) or vector control. Cells were co-cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye. A) Representative example fluorescent imaging of the indicated co-cultures showing the presence of EGFP/mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs. Scale bar indicates 50 μm. B) Quantification of nuclei per EGFP/mCherry double positive cell syncytia in co-cultures involving the indicated IFITM3 constructs as compared to vector control. Bars depict averages of three independent experiments with circles representing the individual data points. For each condition, nuclei within 50-75 individual syncytia were counted. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


HEK293T - ACE2 - GFP stable were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h . B ) Graph depicts normalized infection measurements from 3 independent experiments , each performed in triplicate , from infected as in ( B ) . Bars represent averages with data points shown as circles . Error bars represent SD . blue dotted is added to graph to assist with comparisons to vector control of infection . # p < 0 . 05 by ANOVA followed by Tukey ' s multiple comparisons . NS , not significant . Not indicated , additional comparisons of interest with p < 0 . 05 include IFITM3 ( - ) vs . IFITM3 - Y20 ( - ) , IFITM3 ∆ 59 - 68 ( - ) , IFITM3 - S61A , N64A , T65A ( - ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h. B) Graph depicts normalized infection percentage measurements from 3 independent experiments, each performed in triplicate, from cells infected as in (B). Bars represent averages with individual data points shown as circles. Error bars represent SD. The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection. #p<0.05 by ANOVA followed by Tukey's multiple comparisons test. NS, not significant. Not indicated, additional comparisons of interest with p<0.05 include IFITM3 (-) vs. IFITM3-Y20 (-), IFITM3∆59-68 (-), and IFITM3-S61A,N64A,T65A (-).


B Immunofluorescent confirmed absence of MeCP2 in MECP2 - KO PSCs , NPCs , neurons . Scale bar = 10 µm .

O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O

B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2-KO PSCs, NPCs, and neurons. Scale bar = 10 µm.


C RT - qPCR array revealed differential gene expression between control MECP2 - KO 28 - day neurons . P values are calculated on a Student ' s t - of replicate 2 - ΔΔCt values for each gene in control group KO groups ( WT83 / Q83X lines were used ; N = 3 clones from each genotype ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C RT-qPCR array revealed differential gene expression between control and MECP2-KO 28-day neurons. The P values are calculated based on a Student's t-test of the replicate 2-ΔΔCt values for each gene in the control group and KO groups (WT83/Q83X cell lines were used; N = 3 clones from each genotype).


D Overtime quantitative gene expression profile of many of involved genes relevant to synaptic function ( days 14 28 post differentiation ; normalized by control NPC expression ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function (days 14 and 28 post differentiation; normalized by control NPC expression).


E Single - RT - qPCR distinguished populations of control KO 28 - day neurons . Principal Component ( PCA ) of 441 projected onto first two components . Overlaid populations of WT83 / Q83X WT82 / K82fs neurons are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Single-cell RT-qPCR analysis distinguished populations of control and KO 28-day neurons. Principal Component Analysis (PCA) of 441 cells projected onto the first two components. Overlaid populations of WT83/Q83X and WT82/K82fs neurons are shown.


F Violin plots of selected genes showing comparison between control MECP2 - KO neurons from single - analyses ( log2 ( expression ) values ) that overlapped with results obtained via RT - PCR array .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

F Violin plots of selected genes showing the comparison between control and MECP2-KO neurons from the single-cell analyses (log2(expression) values) that overlapped with the results obtained via RT-PCR array.


G Volcano plot illustrates differences in expression patterns of target genes of neurons from qPCR single - analyses . dotted lines represent differentially expressed genes between groups at P < 0 . 05 ( unpaired Student ' s t - ) .

G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single-cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student's t-test).


H Left : Schematic of laminar cortical markers . Right : Top : Quantitation of laminar markers on single - RT - qPCR ( two - way of variance ( ANOVA ) , N = 3 replicates per genotype , F3 , 24 = 3 . 31 , * P = 0 . 04 , with difference in proportion of cortical layers V VI , 95 % confidence interval ( CI ) [ 8 . 31 , 42 . 41 ] ) ; Bottom : altered proportions of neurotransmitter markers ( two - way ANOVA , N = 3 replicates per genotype , F5 , 24 = 27 . 33 , * * * P < 0 . 0001 , with differences in proportions of glutamatergic , 95 % CI [ 22 . 33 , 33 . 60 ] , cholinergic , 95 % CI [ 0 . 166 , 11 . 43 ] , neurons ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Left: Schematic of laminar cortical markers. Right: Top: Quantitation of laminar cell markers based on the single-cell RT-qPCR analysis (two-way analysis of variance (ANOVA), N = 3 replicates per genotype, F3,24 = 3.31, *P = 0.04, with difference in the proportion of cortical layers V and VI, 95% confidence interval (CI) [8.31, 42.41]); Bottom: altered proportions of neurotransmitter markers (two-way ANOVA, N = 3 replicates per genotype, F5,24 = 27.33, ***P < 0.0001, with differences in the proportions of glutamatergic, 95% CI [22.33, 33.60], and cholinergic, 95% CI [0.166, 11.43], neurons).


D , E Western blot showed decreased presynaptic Synapsin1 ( D ) postsynaptic PSD - 95 ( E ) in untreated 6 - week MECP2 - KO neurons that can be increased by drug treatment ( Kruskal - Wallis , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; full Western blot , Fig EV4A ) ( WT83 / Q83X lines were used ; N = 3 clones from each genotype ) .

O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E Western blot quantification showed decreased presynaptic Synapsin1 (D) and postsynaptic PSD-95 (E) in untreated 6-week MECP2-KO neurons that can be increased by drug treatment (Kruskal-Wallis test, *P < 0.05, **P < 0.01, ***P < 0.001; full Western blot, Fig EV4A) (WT83/Q83X cell lines were used; N = 3 clones from each genotype).


F , G 6 - week MECP2 - KO neurons ( F ) showed a pharmacologically rescuable reduction of co - localized synaptic ( G ; one - way ANOVA , F16 , 118 = 9 . 148 , * P < 0 . 05 ; Dunnet ' s multiple comparisons vs untreated KO ( WT83 / Q83X lines were used ; N = 7 - 8 neurons / condition ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Z scores relative to KO untreated : Control = 3 . 903 ; Nefiracetam = 3 . 560 ; Carbamoylcholine = 2 . 705 ; Pirenzepine = 0 . 0448 ; PHA543613 = 2 . 121 ; Acamprosate = 3 . 339 ; Baclofen = 0 . 5672 ; GR73632 = 2 . 237 ; Hyperforin = 3 . 016 , IGF - 1 = 3 . 791 . Scale bar = 5 µm .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O

F, G 6-week MECP2-KO neurons (F) showed a pharmacologically rescuable reduction of co-localized synaptic puncta (G; one-way ANOVA, F16,118 = 9.148, *P < 0.05; Dunnet's multiple comparisons test vs untreated KO (WT83/Q83X cell lines were used; N = 7-8 neurons/condition). *P < 0.05, **P < 0.01, ***P < 0.001. Z scores relative to KO untreated: Control = 3.903; Nefiracetam = 3.560; Carbamoylcholine = 2.705; Pirenzepine = 0.0448; PHA543613 = 2.121; Acamprosate = 3.339; Baclofen = 0.5672; GR73632 = 2.237; Hyperforin = 3.016, and IGF-1 = 3.791. Scale bar = 5 µm.


H Drug treatment increased calcium transient frequency in 6 - week neurons . changes reflecting intracellular calcium transients in neurons in different regions of interest ( one - way ANOVA , F4 , 52 = 20 . 28 , Dunnett ' s multiple comparisons vs untreated KO : Nefiracetam , * * P < 0 . 01 Z = 2 . 364 ; PHA 543613 , * * * P < 0 . 001 Z = 3 . 391 ; Acamprosate , * * * P < 0 . 001 Z = 3 . 153 ; WT83 / Q83X lines were used ; N = 10 - 14 neurons / condition ) of active neurons ( one - way ANOVA , F4 , 52 = 23 . 11 ; Dunnett ' s multiple comparison vs untreated KO : Nefiracetam , * * * P < 0 . 001 Z = 3 . 144 ; PHA 543613 , * * P < 0 . 01 Z = 2 . 567 ; * * Acamprosate , P < 0 . 01 Z = 2 . 285 ; N = 10 - 14 neurons / condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H Drug treatment increased calcium transient frequency in 6-week neurons. Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest (one-way ANOVA, F4,52 = 20.28, Dunnett's multiple comparisons test vs untreated KO: Nefiracetam, **P < 0.01 and Z = 2.364; PHA 543613, ***P < 0.001 and Z = 3.391; Acamprosate, ***P < 0.001 and Z = 3.153; WT83/Q83X cell lines were used; N = 10-14 neurons/condition) and the percentage of active neurons (one-way ANOVA, F4,52 = 23.11; Dunnett's multiple comparison test vs untreated KO: Nefiracetam, ***P < 0.001 and Z = 3.144; PHA 543613, **P < 0.01 and Z = 2.567; **Acamprosate, P < 0.01 and Z = 2.285; N = 10-14 neurons/condition).


I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2 - KO neurons on MEA ( two - sided unpaired Student ' s t - compared to KO untreated : Nefiracetam , * P = 0 . 016 Z = 2 . 418 ; PHA 543613 , * P = 0 . 027 Z = 2 . 147 ; Acamprosate , P = 0 . 39 Z = 0 . 477 ; WT83 / Q83X WT82 / K82fs lines were used ; N = 4 - 11 MEA wells / condition ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2-KO neurons on MEA (two-sided unpaired Student's t-test compared to KO untreated: Nefiracetam, *P = 0.016 and Z = 2.418; PHA 543613, *P = 0.027 and Z = 2.147; Acamprosate, P = 0.39 and Z = 0.477; WT83/Q83X and WT82/K82fs cell lines were used; N = 4-11 MEA wells/condition).


B , C Immunofluorescent ( B ) quantitation ( C ) confirmed graded decrease in MeCP2 expression from control to 50 % MECP2 mosaic to full MECP2 - KO neurospheres . Scale bar = 50 µm .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O

B, C Immunofluorescent staining (B) and quantitation (C) confirmed the graded decrease in MeCP2 expression from control to 50% MECP2 mosaic to full MECP2-KO neurospheres. Scale bar = 50 µm.


D 8 - week MECP2 - KO neurospheres ( N = 44 ) exhibited decreased ( diameter ; * * P < 0 . 01 ) , but 50 % MECP2 - mosaic neurospheres ( CTR / KO ; N = 44 ) were unchanged from controls ( one - way ANOVA , F2 , 150 = 6 . 03 , P = 0 . 003 ; Dunnett ' s multiple comparisons ; N = 65 ) . Left : representative brightfield of neurospheres ; Right : neurosphere . Scale bar = 200 µm .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 8-week MECP2-KO neurospheres (N = 44) exhibited decreased size (diameter; **P < 0.01), but 50% MECP2-mosaic neurospheres (CTR/KO; N = 44) were unchanged from controls (one-way ANOVA, F2,150 = 6.03, P = 0.003; Dunnett's multiple comparisons test; N = 65). Left: representative brightfield images of neurospheres; Right: neurosphere size quantification. Scale bar = 200 µm.


E Calcium imaging showing calcium transients normalized by control ( one - way ANOVA , F2 , 72 = 15 . 62 , P < 0 . 0001 ; Dunnett ' s multiple comparisons vs control : MECP2 - mosaic , * * * P < 0 . 001 ; MECP2 - KO , * * * P < 0 . 001 ) . WT83 / Q83X lines were used ; N = 44 Q83X neurospheres 65 WT83 neurospheres .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Calcium imaging analysis showing calcium transients normalized by control (one-way ANOVA, F2,72 = 15.62, P < 0.0001; Dunnett's multiple comparisons test vs control: MECP2-mosaic, ***P < 0.001; MECP2-KO, ***P < 0.001). WT83/Q83X cell lines were used; N = 44 Q83X neurospheres and 65 WT83 neurospheres.


F 8 - week neurosphere ( diameter ) remained unchanged by treatment with either drug ( one - way ANOVA , F6 , 344 = 0 . 52 , P = 0 . 79 ; N = 44 Q83X neurospheres 65 WT83 neurospheres ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F 8-week neurosphere size (diameter) remained unchanged by treatment with either drug (one-way ANOVA, F6,344 = 0.52, P = 0.79; N = 44 Q83X neurospheres and 65 WT83 neurospheres).


G viability was improved or unharmed in treated 8 - week neurospheres ( one - way ANOVA , F9 , 104 = 6 . 81 ; Dunnett ' s multiple comparisons vs CTR / KO untreated : Nefi + PHA 1 µM , * * P < 0 . 01 Z = 2 . 974 ; Nefi + PHA 10 µM , * * * P < 0 . 001 Z = 3 . 733 ; N = 8 - 24 neurospheres per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Cell viability was improved or unharmed in treated 8-week neurospheres (one-way ANOVA, F9,104 = 6.81; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi + PHA 1 µM, **P < 0.01 and Z = 2.974; Nefi + PHA 10 µM, ***P < 0.001 and Z = 3.733; N = 8-24 neurospheres per condition).


H , I Calcium transient frequency ( number of peaks in 10 mins recording ) normalized by CTR / KO untreated ( H ; one - way ANOVA , F9 , 239 = 6 . 18 ; Dunnett ' s multiple comparisons vs CTR / KO untreated : Nefi 1 µM , * P < 0 . 05 Z = 0 . 780 ; Nefi + PHA 10 µM , * * * P < 0 . 001 Z = 1 . 972 ; N = 15 - 61 neurospheres per condition ) calcium transient amplitude normalized by CTR / KO untreated ( I ; one - way ANOVA , F9 , 241 = 1 . 27 , P = 0 . 255 ; N = 16 - 62 neurospheres per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H, I Calcium transient frequency (number of peaks in 10 mins recording) normalized by CTR/KO untreated (H; one-way ANOVA, F9,239 = 6.18; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi 1 µM, *P < 0.05 and Z = 0.780; Nefi + PHA 10 µM, ***P < 0.001 and Z = 1.972; N = 15-61 neurospheres per condition) and calcium transient amplitude normalized by CTR/KO untreated (I; one-way ANOVA, F9,241 = 1.27, P = 0.255; N = 16-62 neurospheres per condition).


J , K MEA spike frequency ( J ) ( K ) normalized by CTR / KO untreated ( one - way ANOVA , F5 , 38 = 1 . 21 , P = 0 . 321 ; Dunnett ' s multiple comparisons vs CTR / KO untreated : PHA , P = 0 . 65 Z = 1 . 134 ; Nefi , P = 0 . 98 Z = 0 . 06 ; PHA + Nefi , P = 0 . 86 Z = 0 . 94 ; N = 7 - 8 wells per condition ) . ' X ' on activity signifies absence of a neurosphere in that position .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, K MEA spike frequency heatmap (J) and quantification (K) normalized by CTR/KO untreated (one-way ANOVA, F5,38 = 1.21, P = 0.321; Dunnett's multiple comparisons test vs CTR/KO untreated: PHA, P = 0.65 and Z = 1.134; Nefi, P = 0.98 and Z = 0.06; PHA + Nefi, P = 0.86 and Z = 0.94; N = 7-8 wells per condition). 'X' on activity heatmap signifies absence of a neurosphere in that position.


B 2 - month old cortical organoid diameter ( one - way ANOVA , F2 , 173 = 91 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons vs untreated : Nefiracetam , * * * P < 0 . 001 Z = 5 . 414 ; PHA 543613 , * * * P < 0 . 001 Z = 5 . 351 ; WT83 / Q83X WT82 / K82fs lines were used ; N = 28 - 90 organoids per condition ) . Left : representative brightfield of cortical organoids ; Right : cortical organoid . Scale bar = 200 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B 2-month old cortical organoid diameter (one-way ANOVA, F2,173 = 91.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, ***P < 0.001 and Z = 5.414; PHA 543613, ***P < 0.001 and Z = 5.351; WT83/Q83X and WT82/K82fs cell lines were used; N = 28-90 organoids per condition). Left: representative brightfield images of cortical organoids; Right: cortical organoid size quantification. Scale bar = 200 µm.


C Ki67 + proliferative ( Student ' s t - , t10 = 0 . 36 , P = 0 . 73 , N = 6 each ) . Left : representative immunofluorescence of progenitor , proliferative , neuronal markers in cortical organoids ; Right : of Ki67 + proliferation in cortical organoids . Scale bar = 50 µm .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

C Ki67+ proliferative area (Student's t-test, t10 = 0.36, P = 0.73, N = 6 each). Left: representative immunofluorescence images of progenitor, proliferative, and neuronal markers in cortical organoids; Right: quantification of Ki67+ proliferation in cortical organoids. Scale bar = 50 µm.


sequencing ( D , left : ) GO ( D , right ) of untreated MECP2 - KO organoids vs Nefiracetam ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated genetic expression in synapse - relevant pathways .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA sequencing (D, left: heatmap) and GO analysis (D, right) of untreated MECP2-KO organoids vs Nefiracetam (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated genetic expression in synapse-relevant pathways.


E , center band of box is median log2 normalized expression edges are 25th 75th percentiles ; whiskers are minimum maximum log2 normalized expression values .

O O O O O O O O O O O O O O O O O O O O O O O O O O

E, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.


sequencing ( F , left : ) GO ( F , right ) of untreated MECP2 - KO organoids vs PHA 543613 ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated expression of genes in synapse - relevant pathways .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNA sequencing (F, left: heatmap) and GO analysis (F, right) of untreated MECP2-KO organoids vs PHA 543613 (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated expression of genes in synapse-relevant pathways.


G , center band of box is median log2 normalized expression edges are 25th 75th percentiles ; whiskers are minimum maximum log2 normalized expression values .

G, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.


H Synapsin1 + quantitation ( one - way ANOVA , F3 , 34 = 10 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons vs untreated : Nefiracetam , * * P < 0 . 01 Z = 2 . 998 ; PHA 543613 , P > 0 . 05 Z = 1 . 266 ; WT83 / Q83X lines were used ; N = 8 - 10 each ) . Left : representative image of Synapsin1 + immunofluorescent ; Right : Synapsin1 + . Scale bar = 20 µm .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O

H Synapsin1+ puncta quantitation (one-way ANOVA, F3,34 = 10.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, **P < 0.01 and Z = 2.998; PHA 543613, P > 0.05 and Z = 1.266; WT83/Q83X cell lines were used; N = 8-10 each). Left: representative image of Synapsin1+ immunofluorescent staining; Right: Synapsin1+ puncta quantification. Scale bar = 20 µm.


MECP2 - KO control organoids had proportions of positive for neuronal marker NeuN ( I ; Student ' s t - , t14 = 0 . 26 , P = 0 . 80 , N = 8 each )

B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

MECP2-KO and control organoids had proportions of cells positive for the neuronal marker NeuN (I; Student's t-test, t14 = 0.26, P = 0.80, N = 8 each)


J MECP2 - KO control organoids had proportions of positive for cortical layer V VI neuronal marker CTIP2 ( J ; one - way ANOVA , F3 , 40 = 2 . 489 , P = 0 . 074 Z < 1 , WT83 / Q83X WT82 / K82fs lines were used ; N = 8 - 14 organoids each ) . J , left : representative image of CTIP2 + immunofluorescent in cortical organoids ; J , right : of CTIP2 + neurons . Scale bar = 50 µm .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

J MECP2-KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 (J; one-way ANOVA, F3,40 = 2.489, P = 0.074 and Z < 1, WT83/Q83X and WT82/K82fs cell lines were used; N = 8-14 organoids each). J, left: representative image of CTIP2+ immunofluorescent staining in cortical organoids; J, right: quantification of CTIP2+ neurons. Scale bar = 50 µm.