( a ) Mass spectrometry of co - immunoprecipitating with eIF4A reveals NAT1 as one of top interacting . eIF4A immunoprecipitations were performed in triplicate , from treated with medium either containing ( + aa ) or lacking ( - aa ) , average values are shown . Peptide for each in each replicate were normalized to eIF4A peptide . Raw peptide shown in parentheses . ( a ' ) Schematic diagram of eIF4G NAT1 structures . Binding sites for other initiation factors are shown .

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(a) Mass spectrometry analysis of proteins co-immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins. eIF4A immunoprecipitations were performed in triplicate, from cells treated with medium either containing (+aa) or lacking amino acids (-aa), and average values are shown. Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts. Raw peptide counts shown in parentheses.(a') Schematic diagram of eIF4G and NAT1 primary protein structures. Binding sites for other initiation factors are shown.


( b ) Binding between eIF4A NAT1 is regulated by acid availability in a TORC1 - independent fashion . Co - immunoprecipitation of tagged eIF4A NAT1 in control Kc167 , or treated with medium lacking or supplemented with 20 nM rapamycin for indicated times . Representative of 3 biological replicates .

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(b) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1-independent fashion. Co-immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times. Representative of 3 biological replicates.


( c ) Knockdown of NAT1 using 4 independent , non - overlapping dsRNAs leads to reduced TORC1 activity . Kc167 , treated with indicated dsRNAs for 4 days then incubated with medium containing or lacking for 30 min . Error bars : std . dev . n = 3 biological replicates .

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(c) Knockdown of NAT1 using 4 independent, non-overlapping dsRNAs leads to reduced TORC1 activity. Kc167 cells, treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.


( d ) eIF4A is epistatic to NAT1 for TORC1 regulation . In absence of , eIF4A knockdown have elevated TORC1 activity , NAT1 knockdown have reduced TORC1 activity , eIF4A NAT1 double - knockdown have elevated TORC1 activity compared to control . were treated with indicated dsRNAs for 4 days then treated with medium containing or lacking indicated for 30 min . Error bars : std . dev . n = 3 biological replicates .

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(d) eIF4A is epistatic to NAT1 for TORC1 regulation. In the absence of amino acids, eIF4A knockdown cells have elevated TORC1 activity, NAT1 knockdown cells have reduced TORC1 activity, and eIF4A NAT1 double-knockdown cells have elevated TORC1 activity compared to control cells. Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.


( e ) TSC2 is epistatic to NAT1 for TORC1 regulation . As in ( d ) , knockdown of TSC2 rescues reduced TORC1 activity caused by NAT1 knockdown .

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(e) TSC2 is epistatic to NAT1 for TORC1 regulation. As in (d), knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown.


A Anti - TK Western blot of ubiquitinated titin samples .

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A Anti-TK Western blot of ubiquitinated titin samples.


( B - D ) of H9C2 corresponding to ( B ) expressions of single species : MuRF1 - mCherry ( red ) , titin EGFP - A168 - TK ( green ) , Venus - Nbr1 ( yellow ) or Cerulean - p62 ( cyan ) ; ( C ) Co - expression of titin EGFP - A168 - TK with mCherry - MuRF1 . Neither Nbr1 nor p62 were co - transfected . titin fragment MuRF1 co - localize forming filamentous formations , which is in agreement with their known complexation ; ( D ) Co - expression of MuRF1 - mCherry ( red ) , titin EGFP - A168 - TK ( green ) Venus - Nbr1 ( yellow ) or Cerulean - p62 ( cyan ) or respective truncated variants of latter lacking ubiquitin binding domain ( ∆ UBA ) . ( Throughout , nuclei are stained with DAPI ; blue ) . ( Scale bar is 10 μm in cases ) .

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(B-D) Micrographs of H9C2 cells corresponding to (B) expressions of single species: MuRF1-mCherry (red), titin EGFP-A168-TK (green), Venus-Nbr1 (yellow) or Cerulean-p62 (cyan); (C) Co-expression of titin EGFP-A168-TK with mCherry-MuRF1. Neither Nbr1 nor p62 were co-transfected. The titin fragment and MuRF1 co-localize forming filamentous formations, which is in agreement with their known complexation; (D) Co-expression of MuRF1-mCherry (red), titin EGFP-A168-TK (green) and Venus-Nbr1 (yellow) or Cerulean-p62 (cyan) or respective truncated variants of the latter lacking the ubiquitin binding domain (∆UBA). (Throughout, cell nuclei are stained with DAPI; blue). (Scale bar is 10 μm in all cases).


Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( A ) Induced Cyp1a1 mRNA expression in response to different treatments ( n = 3 per group ) .

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Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (A) Induced Cyp1a1 mRNA expression in response to different treatments (n=3 per group).


Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( B ) Assessment of GFP + percentages ( n = 3 replicates per treatment ) . Bars with different letters are significantly different ( p < 0 . 05 ) .

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Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (B) Assessment of GFP+ stem cell percentages (n=3 replicates per treatment). Bars with different letters are significantly different (p<0.05).


( D ) Flow cytometry of GFP + Tomato + Tomato + in large intestine of AhR wild type ( Lgr5 - GFPCreERT2 X Tomatof / f , GT ) AhR knock out ( AhRf / f X Lgr5 - GFPCreERT2 X Tomatof / f , HGT ) mice . Top left panel shows representative from WT negative control ( no GFP or Tomato expression ) . Top right panel shows Tomato only control ( CDX2P - CreERT2 X Tomatof / f ) . Middle panels represent DMSO or TCDD treatment in AhR WT mice , respectively . Mice were gavaged every other day , 4 times total , with vehicle or TCDD ( 25 µg / kg bw ) terminated 1 d later . Bottom left panel is from a representative AhR WT mouse bottom right panel is from a representative AhR KO mouse .

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(D) Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type (Lgr5-GFPCreERT2 X Tomatof/f, GT) and AhR knock out (AhRf/f X Lgr5-GFPCreERT2 X Tomatof/f, HGT) mice. Top left panel shows representative cells from WT negative control animal (no GFP or Tomato expression). Top right panel shows Tomato only control (CDX2P-CreERT2 X Tomatof/f). Middle panels represent DMSO or TCDD treatment in AhR WT mice, respectively. Mice were gavaged every other day, 4 times total, with vehicle or TCDD (25 µg/kg bw) and terminated 1 d later. Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse.


( E ) Induced Cyp1a1 mRNA expression in after oral gavage with TCDD ( n = 4 ) .

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(E) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD (n=4).


( F ) of Lgr5 + ( n = 4 mice per group ) in ( D ) .

(F) Percentage of Lgr5+ stem cells (n=4 mice per group) in (D).


Flow cytometry of GFP + Tomato + Tomato + in large intestine of AhR wild type AhR knock out mice . ( G ) Quantitative of of Lgr5 + in ( D ) , n = 7 - 10 mice per group .

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Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type and AhR knock out mice. (G) Quantitative analysis of the percentage of Lgr5+ stem cells in (D), n=7-10 mice per group.


Representative immunohistochemistry of GFP + per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm .

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Representative immunohistochemistry of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm.


Representative violin plots of GFP + per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm . Solid indicates median , dotted lines indicate 25th / 75th quartiles .

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Representative violin plots of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm. Solid line indicates median, and dotted lines indicate 25th/75th quartiles.


( A ) Representative flow cytometry gating microscopy of sorted GFPhigh GFPlow progenitor from Lgr5 - EGFP - IRES - CreERT2 mice .

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(A) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5-EGFP-IRES-CreERT2 mice.


( B ) Representative brightfield of organoids generated from GFPhigh isolated from tamoxifen treated AhR WT mice ( Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ GC ] ) KO mice ( AhRf / f X Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ HGC ] ) , treated with DMSO ( control ) or TCDD for 5 d . Data are from ≥ 4 mice , scale bar 200 µm .

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(B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm.


( C , D ) organoid forming efficiency ( C ) diameter ( D ) from sorted GFPhigh in AhR WT or KO mice treated for 5 d with DMSO or TCDD ( 10 nM ) . n = 4 - 5 mice per group in ( C ) . Each dot represents an organoid grey indicates mean in ( D ) . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05).


Organoid growth from GFPlow progenitor . ( E ) Representative brightfield of organoids .

Organoid growth from GFPlow progenitor cells. (E) Representative brightfield images of organoids.


( F ) Organoid forming efficiency ( G ) diameter . n = 4 - 8 mice per group in ( F ) . Each dot represents an organoid grey indicates mean in ( G ) . Scale bar 200 μm . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(F) Organoid forming efficiency and (G) diameter. n=4-8 mice per group in (F). Each dot represents an individual organoid and grey line indicates mean in (G). Scale bar 200 μm. Bars with different letters are significantly different (p<0.05).


( H , I ) ( H ) Lgr5 expression in organoids ( I ) sorted in absence or presence of TCDD treatment ( n ≥ 3 independent samples per group ) .

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(H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group).


( J ) Representative brightfield for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d . Scale 200 µm .

(J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.


( C ) comparison of top 25 upstream regulators between AhR KO ( KO / WT ) TCDD ( TCDD / vehicle in WT mice ) treated .

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(C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells.


( A ) FoxM1 , Plk1 , Cdc25B mRNA expression in progenitor with without AhR , n = 4 mice per group .

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(A) FoxM1, Plk1, and Cdc25B mRNA expression in stem and progenitor cells with and without AhR, n=4 mice per group.


( B ) FoxM1 , Plk1 , Cdc25B mRNA expression in WT progenitor with without TCDD , n = 3 - 4 mice per group .

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(B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group.


immunoprecipitation ( ChIP ) of AhR FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD , n = 3 or 4 per group . A fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control .

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Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control.


immunoprecipitation ( ChIP ) of AhR FoxM1 promoter interaction using WT KO crypts ( E ) treated with DMSO or TCDD , n = 3 or 4 per group .

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Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group.


( F ) FoxM1 luciferase activity determination for young adult mouse colonocytes ( YAMCs ) treated with DMSO or 10 nM TCDD for 1 d , n = 3 per group .

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(F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group.


( G Expression of FoxM1 PLK1 at mRNA level , , n > = 3 independent organoids per treatment .

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(G Expression of FoxM1 and PLK1 at mRNA level, , n>=3 independent organoids per treatment.


H ) Expression of FoxM1 PLK1 at level , n > = 3 independent organoids per treatment .

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H) Expression of FoxM1 and PLK1 at protein level, n>=3 independent organoids per treatment.


( I ) Representative brightfield of mouse colonic organoids generated from WT Lgr5 + - GFPhi treated with DMSO or 5µM FDI - 6 . Scale bar 200 µm .

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(I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm.


( J , K ) of organoid forming efficiency ( J ) organoid ( K ) from WT colonic treated with DMSO or 5µM FDI - 6 . Data are represented as mean ± in ( K ) .

(J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K).


( L ) Organoid expression of Lgr5 in response to FDI - 6 . Lgr5 expression level was dramatically suppressed by FDI - 6 in a dose - dependent manner , n > = 3 per treatment .

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(L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment.


( M ) Representative brightfield for secondary organoid formation following treatment with DMSO or 5 µM FDI - 6 for 2 d . Scale 200 µm .

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(M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.


( A B ) Proliferating Lgr5 + marked by EdU in distal colon from AhR WT ( GT ) KO ( HGT ) mice . Arrowhead star symbols denote proliferating ( GFP + EdU + ) static ( GFP + EdU - ) , respectively , during a 2 h EdU , n = 3 per treatment .

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(A and B) Proliferating Lgr5+ stem cells marked by EdU in the distal colon from AhR WT (GT) and KO (HGT) mice. Arrowhead and star symbols denote proliferating stem cells (GFP+EdU+) and static stem cells (GFP+EdU-), respectively, during a 2 h EdU pulse, n=3 per treatment.


( C ) Total number of EdU + per crypt in distal colon of AhR WT KO ( HGT ) mice , n = 3 mice per treatment . Dashed indicates median dotted lines indicate 25th / 75th quartiles .

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(C) Total number of EdU+ cells per crypt in the distal colon of AhR WT and KO (HGT) mice, n=3 mice per treatment. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( D & E ) Crypt proliferative zone in AhR WT KO ( HGT ) mice , presented as from crypt base to highest EdU + divided by total crypt . Red lines between crosses show measurement of . Each symbol represents one crypt , n = 3 mice per treatment , scale bar 20 µm . Grey represents mean .

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(D&E) Crypt cell proliferative zone analysis in AhR WT and KO (HGT) mice, presented as the distance from the crypt base to the highest EdU+ cell divided by total crypt length. Red lines between crosses show the measurement of distance. Each symbol represents one crypt, n=3 mice per treatment, scale bar 20 µm. Grey line represents mean.


( F & AhR KO promoted proliferation 5 d post DSS treatment . AhR WT ( AhRf / f [ H ] , n = 4 ) AhR KO ( AhRf / f X Villin - Cre [ HV ] , n = 4 ) mice were used .

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(F& AhR KO promoted cell proliferation 5 d post DSS treatment. AhR WT (AhRf/f [H], n=4) and AhR KO (AhRf/f X Villin-Cre [HV], n=4) mice were used.


G ) AhR KO promoted proliferation 5 d post DSS treatment . Data represent mean ± , quantified as of EdU + relative to DAPI + using KeyenceTM software .

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G) AhR KO promoted cell proliferation 5 d post DSS treatment. Data represent mean ± SEM, quantified as the ratio of the EdU+ area relative to the DAPI+ area using KeyenceTM software.


( H ) Increased proliferation ( EdU + ) observed in AhR KO vs WT organoids derived from HGC GC mice , respectively ( n = 6 from 3 mice per group ) .

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(H) Increased cell proliferation (EdU+ cells) observed in AhR KO vs WT organoids derived from HGC and GC mice, respectively (n=6 from 3 mice per group).


( I ) Effect of AhR KO on cycle S G2 - M phases ( n = 3 mice per group ) .

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(I) Effect of AhR KO on cell cycle S and G2-M phases (n=3 mice per group).


( J ) . FoxM1 inhibition reduced number of colonic proliferation . Mice were intraperitoneally injected with FoxM1 inhibitor Thiostrepton ( 50 mg / kg ) for 2 consecutive days , n = 80 ~ 100 crypts from 4 mice per group . Scale bar , 50 µm . Dashed indicates median dotted lines indicate 25th / 75th quartiles . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(J). FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation. Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton (50 mg/kg) for 2 consecutive days, n=80~100 crypts from 4 mice per group. Scale bar, 50 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. Bars with different letters are significantly different (p<0.05).


( B ) Representative colon from AhR WT AhR KO mice following AOM / DSS treatment . Scale bar , 9 mm .

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(B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm.


( C ) H & E of representative colon sections from AhR WT KO mice , scale bar 200 μm .

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(C) H&E staining of representative colon tumor sections from AhR WT and KO mice, scale bar 200 μm.


( E , F ) ( E ) Adenoma ( F ) adenocarcinoma multiplicity in AhR WT KO mice .

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(E, F) (E) Adenoma and (F) adenocarcinoma multiplicity in AhR WT and KO mice.


( G ) volume in AhR KO WT mice . Grey indicates mean .

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(G) Tumor volume in AhR KO and WT mice. Grey line indicates mean.


( H ) FoxM1 mRNA expression in normal ( uninvolved ) crypts adjacent to colonic tumors . were quantified expressed as FoxM1 positive per crypt ( n = 5 mice per group ) . Scale 200 µm . Dashed indicates median dotted lines indicate 25th / 75th quartiles .

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(H) FoxM1 mRNA expression in normal (uninvolved) crypts adjacent to colonic tumors. Images were quantified and expressed as FoxM1 positive area per crypt (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( I ) FoxM1 mRNA expression in colon tumors . FoxM1 mRNA ISH was averaged per on each slide ( n = 5 mice per group ) . Scale 200 µm . Dashed indicates median dotted lines indicate 25th / 75th quartiles .

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(I) FoxM1 mRNA expression in colon tumors. FoxM1 mRNA ISH area was averaged per tumor on each slide (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( A - B ) mRNA expression of human colonic markers LGR5 OLFM4 , n = 5 or 6 independent human samples . Paired student t - .

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(A-B) mRNA expression of human colonic stem cell markers LGR5 and OLFM4, n=5 or 6 independent human samples. Paired student t-test.


( C ) Representative of LGR5 ISH in human organoids treated with DMSO or TCDD . Scale bar 50 µm .

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(C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm.


mRNA expression of target genes , n = 5 or 6 independent human samples . Paired student t - .

mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.


( H ) Representative immunoblots for FoxM1 PLK1 derived from organoids treated with DMSO or TCDD from n = 6 independent samples . Total was used as a loading control .

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(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.


( I ) Determination of luciferase reporter activity of human FoxM1 in SW48 Caco2 lines treated with DMSO or TCDD . n = 3 - 4 per group .

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(I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group.


( J ) FOXM1 CYP1A1 mRNA expression in different AhR WT CRISPR KO clones treated with TCDD for 1 d . C56 C61 designate different human subjects .

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(J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects.


( K ) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking targeted AhR exon . Subsequent sequencing revealed indels at expected locations .

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(K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations.


( L ) association of FoxM1 AhR mRNA expression in human normal colon biopsies ( n = 304 ) . AhR low ( n = 152 ) is defined as below its median expression ; AhR high ( n = 152 ) is defined as above its median expression . Dashed indicates median dotted lines indicate 25th / 75th quartiles .

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(L) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies (n=304). AhR low (n=152) is defined as below its median expression; AhR high (n=152) is defined as above its median expression. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( M ) Correlation between FoxM1 Cyp1b1 mRNA expression in human normal colon biopsies ( n = 303 ) analyzed by Spearman rank correlation .

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(M) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies (n=303) analyzed by Spearman rank correlation.


( N ) Representative brightfield of human colonic organoids from sorted live at day 6 , scale bar 200 μm .

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(N) Representative brightfield images of human colonic organoids from sorted live cells at day 6, scale bar 200 μm.


( O , P ) ( O ) Human organoid forming efficiency , ( P ) organoid diameter from sorted from human colonic organoids in presence of DMSO , TCDD CH223191 ( 10 μM ) for 6 d . n = 5 per group , 3 independent experiments . Grey indicates mean in ( P ) . Treatments not sharing a common letter are significantly different , p < 0 . 05 .

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(O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05.


( Q ) Representative brightfield of human colonic organoids treated with DMSO or FDI - 6 . FoxM1 inhibition suppressed human colonic organoid growth in a dose - dependent manner , scale bar 200 µm .

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(Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.


Survival , gain of mobility plots for Armadillidium vulgare during 75 days post - injection . There were no significant differences between injected with wVulC , wDil or control treatment for these life history traits : Survival ( A ) , Gain of ( B ) Mobility ( C ) .

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Survival, gain of weight and mobility plots for Armadillidium vulgare during 75 days post-injection.There were no significant differences between animals injected with wVulC, wDil or control treatment for all these life history traits: Survival (A), Gain of weight (B) and Mobility (C).


were injected by pure suspension of wVulC , diluted wVulC inoculum ( only for ) , wDil or control treatment monitored during 75 days post - injection . ( A ) Gain of : Both Wolbachia strains negatively impact this parameter compared to control treatment . However , wVulC ( i . e pure or 1 / 100 diluted inoculum ) induces a lower growth rate than native

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Animals were injected by pure suspension of wVulC, diluted wVulC inoculum (only for females), wDil or control treatment and monitored during 75 days post-injection. (A) Gain of weight: Both Wolbachia strains negatively impact this parameter compared to the control treatment. However, wVulC (i.e pure or 1/100 diluted inoculum) induces a lower growth rate than the native strain wDil


( B ) Mobility : this parameter is dramatically reduced for injected by wVulC compared to those from other treatments . On opposite , injection of wDil has no significant effect on host ' s mobility .

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(B) Mobility: this parameter is dramatically reduced for animals injected by wVulC compared to those from other treatments. On the opposite, injection of wDil has no significant effect on the host's mobility.


( C ) Burying behaviour : level 0 corresponds to surface of substrate , negative values correspond to number of buried while positive values correspond to number of found at surface of substrate . For control treatment , not a single rose up at surface for individuals injected by wDil only few were spotted at surface . When inoculated by wVulC , rose up to substrate after 60 days .

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(C) Burying behaviour: The level 0 corresponds to the surface of the substrate, negative values correspond to the number of buried animals while positive values correspond to the number of animals found at the surface of the substrate. For control treatment animals, not a single individual rose up at the surface and for individuals injected by wDil only few animals were spotted at the surface. When inoculated by wVulC, all animals rose up to the substrate after 60 days.


( D ) Survival ( a ) data for ( b ) data for : injection of wVulC kills individuals . However , die quicker than . No extra - mortalities were reported for injection with wDil compared to control treatment .

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(D) Survival (a) data for females and (b) data for males: The injection of wVulC kills all the individuals. However, the males die quicker than the females. No extra-mortalities were reported for injection with wDil compared to control treatment.


Wolbachia loads in CNS , gonads hemocytes of Porcellio d . dilatatus Armadillidium vulgare . ( A ) Comparison between ( i ) P . d . dilatatus in grey A . vulgare in injected with wVulC ( ii ) naturally infected A . vulgare in grey revealed no significant differences for Wolbachia loads in CNS , gonads hemocytes ( i . e immune ) , at both 30 60 days post - injection . ( B ) Comparison between ( i ) P . d . dilatatus in grey A . vulgare in injected with wDil ( ii ) naturally infected P . d . dilatatus in dim grey revealed no significant differences for Wolbachia loads in CNS at both 30 60 days PI . However , at 60 days post - injection , for gonads hemocytes wDil loads were significantly higher in native host P . d . dilatatus than in A . vulgare . A global comparison between wDil wVulC loads showed that wVulC ( A ) always exhibited higher loads than wDil ( B ) in both host species .

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Wolbachia loads in CNS, gonads and hemocytes of Porcellio d. dilatatus and Armadillidium vulgare.(A) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wVulC and (ii) naturally infected A. vulgare in light grey revealed no significant differences for the Wolbachia loads in CNS cells, gonads and hemocytes (i.e immune cells), at both 30 and 60 days post-injection. (B) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wDil and (ii) naturally infected P. d. dilatatus in dim grey revealed no significant differences for the Wolbachia loads in CNS cells at both 30 and 60 days PI. However, at 60 days post-injection, for gonads and hemocytes wDil loads were significantly higher in the native host P. d. dilatatus than in A. vulgare. A global comparison between wDil and wVulC loads showed that wVulC (A) always exhibited higher bacterial loads than wDil (B) in both host species.


Micrograph of nerve of Porcellio d . dilatatus injected by wVulC . These are localised in CNS ganglion . ( A ) At 30 days post - injection , some autolysosomes are visible : autophagic process is already quite high compared to control [ N : Nucleus , V : Vacuole , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Wo : Wolbachia , * : a Wolbachia which seems to be surrounded by an autolysosome , Arrow head : magnified ] . ( B ) At 60 days post - injection , nerve are severely damaged by accumulation of autophagic vesicles ( N : Nucleus , V : Vacuole , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Arrow head : magnified probably a Wolbachia in autophagic degradation ) .

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Micrograph of nerve cells of Porcellio d. dilatatus injected by wVulC.These cells are localised in the CNS ganglion. (A) At 30 days post-injection, some autolysosomes and bacteria are visible: autophagic process is already quite high compared to control [N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, *: a Wolbachia which seems to be surrounded by an autolysosome, Arrow head: magnified area]. (B) At 60 days post-injection, the nerve cells are severely damaged by the accumulation of autophagic vesicles (N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Arrow head: magnified area and probably a Wolbachia in autophagic degradation).


Micrograph LC3B antibody labelling of adipocytes neighboring nerve of Porcellio d . dilatatus . These adipocytes are localised around nerve of CNS constitutes sheathing of this organ . ( A ) At 30 days post - injection , autolysosomes , autophagosomes but also were observed : autophagic process was already higher compared to control ( Ad : adipocyte , Ap : Autophagosomes ( Autophagic hallmark ) , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified ) . ( B ) At 60 days post - injection , adipocytes were severely damaged by accumulation of autophagic vesicles ( Ad : adipocyte , Ap : Autophagosomes , Al : Autolysosomes , Wo : Wolbachia , Arrow head : magnified ) . ( C ) With antibody labelling anti - LC3B , LC3B implicated in autophagic processes was detected in adipocytes of P . d . dilatatus injected with wVulC at 45 days post - injection . highly labeled spherical structures would reflect incorporation of LC3B in phagophores . ( D ) With adipocytes of P . d . dilatatus injected by wDil but also in other tested situations , only really few of these structures were detected by epiflorescent microscopy .

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Micrograph and LC3B antibody labelling of adipocytes neighboring the nerve cells of Porcellio d. dilatatus.These adipocytes are localised around the nerve cells of the CNS and constitutes the sheathing of this organ. (A) At 30 days post-injection, autolysosomes, autophagosomes but also bacteria were observed: autophagic process was already higher compared to the control (Ad: adipocyte, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (B) At 60 days post-injection, the adipocytes were severely damaged by the accumulation of autophagic vesicles (Ad: adipocyte, Ap: Autophagosomes, Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (C) With the antibody labelling anti-LC3B, the protein LC3B implicated in autophagic processes was detected in adipocytes of P. d. dilatatus injected with wVulC at 45 days post-injection. The highly labeled spherical structures would reflect the incorporation of the LC3B protein in the phagophores. (D) With adipocytes of P. d. dilatatus injected by wDil but also in other tested situations, only really few of these structures were detected by epiflorescent microscopy.


( A ) Western for on - target effects for Everolimus ( EV , P - RPS6 ) CX - 5461 ( p53 ) . Each lane represents equal amounts of from lymph node tissue isolated from a single mouse that received drug vehicles ( everolimus vehicle : 1 % methylcellulose ; CX - 5461 vehicle : 25 mM NaH2PO 4 ) ( V / V ; mouse # 1 - 6 ) , 5 mg / kg everolimus ( EV ; mouse # 7 - 12 ) , 35 mg / kg CX - 5461 ( mouse # 13 - 18 ) or both drugs ( CX - 5461 + EV ; mouse # 19 - 24 ) for 2 hours ( n = 6 per treatment group ) . Actin was used as a loading control .

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(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX-5461 vehicle: 25 mM NaH2PO4) (V/V; mouse #1-6), 5 mg/kg everolimus (EV; mouse #7-12), 35 mg/kg CX-5461 (mouse #13-18) or both drugs (CX-5461+EV; mouse #19-24) for 2 hours (n=6 per treatment group). Actin was used as a loading control.


( C ) Enrichment by MetaCore ® GeneGO of genes in " translation up " ( red ) " translation down " ( blue ) categories identified by anota2seq comparing lymph node isolated from mice in CX - 5461 + EV treatment group with V / V group ( n = 6 ) . " values were obtained by dividing number of significant genes in our data assigned to a molecular process by total number of genes in process in MetaCore ® ' s database . ( D ) Genes implicated in " Translation : initiation " " Translation : Elongation - Termination " processes on Fig

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(C) Enrichment analysis by MetaCore® GeneGO of genes in "translation up" (red) and "translation down" (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX-5461+EV treatment group with the V/V group (n=6). Ratio" values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore®'s database. (D) Genes implicated in "Translation: initiation" and "Translation: Elongation-Termination" processes based on Fig 1C


( B ) viability of indicated early - passage Eµ - Myc lymphoma treated with indicated compound ( s ) for 48 hours as determined by Beckman ® Coulter counter .

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(B) Cell viability analysis of the indicated early-passage Eµ-Myc lymphoma cells treated with indicated compound(s) for 48 hours as determined by Beckman® Coulter counter.


( E ) Intracellular ATP determined using liquid chromatography - mass spectrometry in CTRL , EV , CX CMB early - passage lines .

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(E) Intracellular ATP levels determined using the liquid chromatography-mass spectrometry in CTRL, EV, CX and CMB early-passage cell lines.


( F ) Basal extracellular acidification rate ( ECAR ) oxygen consumption rate ( OCR ) in early - passage lines determined using Seahorse XF96 Extracellular Flux Analyzer ; graphs represent mean ± of 6 - 8 technical replicates for each biological replicate ( n = 3 ) .

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(F) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the early-passage cell lines determined using the Seahorse XF96 Extracellular Flux Analyzer; graphs represent mean ± SEM of 6-8 technical replicates for each biological replicate (n=3).


( G ) Propidium iodide ( PI ) exclusion of early - passage CX - 5461 - everolimus combination therapy - resistant ( CMB ) , CX - 5461 - resistant ( CX ) drug - naïve ( CTRL ) lymphoma treated with indicated concentrations of metformin for 48 hours .

(G) Propidium iodide (PI) exclusion analysis of early-passage CX-5461-everolimus combination therapy-resistant (CMB), CX-5461-resistant (CX) and drug-naïve (CTRL) lymphoma cells treated with indicated concentrations of metformin for 48 hours.


( H ) CellTiterGLO ® - measuring ATP of CX - 5461 - resistant ( CX ) CX - 5461 - everolimus combination therapy - resistant ( CMB ) treated with metformin as indicated for 48 hours .

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(H) CellTiterGLO®-based assay measuring cellular ATP levels of the CX-5461-resistant (CX) and CX-5461-everolimus combination therapy-resistant (CMB) cells treated with metformin as indicated for 48 hours.


( I ) PI of CTRL CMB treated with CX - 5461 everolimus in presence absence of metformin for 48 hours as indicated .

(I) PI analysis of CTRL and CMB cells treated with CX-5461 and everolimus in the presence and absence of metformin for 48 hours as indicated.


( J ) PI of CTRL CX treated with CX - 5461 in presence absence of metformin for 48 hours as indicated .

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(J) PI analysis of CTRL and CX cells treated with CX-5461 in the presence and absence of metformin for 48 hours as indicated.


( B ) Abundance of intracellular 3 ' 5 ' - cyclic AMP as measured by a liquid chromatography ( LC ) - mass spectrometry ( MS ) . Graphs represent mean ± of n = 3 ( 5 - 6 technical replicates each ) .

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(B) Abundance of intracellular 3'5'-cyclic AMP as measured by a liquid chromatography (LC)-mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n=3 (5-6 technical replicates each).


( C ) Western of EPAC1 EPAC2 abundance , as well as active GTP - bound RAP1 in indicated early - passage during their log - phase growth period ( n = 3 ) . Actin total RAP1 were used as loading controls .

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(C) Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP-bound RAP1 levels in the indicated early-passage cells during their log-phase growth period (n=3). Actin and total RAP1 were used as loading controls.


( D ) Propidium iodide ( PI ) exclusion assays of CMB treated with CX - 5461 everolimus as indicated in presence or absence of a selective PKA inhibitor H89 for 48 hours .

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(D) Propidium iodide (PI) exclusion assays of CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours.


( E ) PI exclusion of early - passage drug - naïve ( CTRL ) lymphoma treated with CX - 5461 everolimus in presence or absence of selective PKA activator 6 - Bnz - cAMP for 48 hours .

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(E) PI exclusion analysis of early-passage drug-naïve (CTRL) lymphoma cells treated with CX-5461 and everolimus in the presence or absence of selective PKA activator 6-Bnz-cAMP for 48 hours.


( F ) PI exclusion of CMB treated with CX - 5461 everolimus as indicated in presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI - 05 for 48 hours .

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(F) PI exclusion analysis of the CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI-05 for 48 hours.


( G ) PI exclusion of early - passage drug - naïve ( CTRL ) treated with CX - 5461 everolimus in presence or absence of selective EPAC activator 8 - pCPT - 2 - O - Me - cAMP for 48 hours .

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(G) PI exclusion analysis of early-passage drug-naïve (CTRL) cells treated with CX-5461 and everolimus in the presence or absence of the selective EPAC activator 8-pCPT-2-O-Me-cAMP for 48 hours.


( A ) A illustrating abundances of polysome - associated mRNAs that are associated with mRNA translation / synthesis on Gene Ontology Resource Database in drug - naïve ( CTRL ) or CX - 5461 - everolimus combination therapy - resistant ( CMB ) lines ( n = 3 ) . colours illustrate expression values of indicated mRNAs that were normalized using voom ( red : high expression ; blue : low expression ) .

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(A) A heatmap illustrating the abundances of polysome-associated mRNAs that are associated with the mRNA translation/protein synthesis based on the Gene Ontology Resource Database in drug-naïve (CTRL) or CX-5461-everolimus combination therapy-resistant (CMB) cell lines (n=3). The colours illustrate the expression values of indicated mRNAs that were normalized using voom (red: high expression; blue: low expression).


( B ) Polysome profiles demonstrating increased ribosome abundance in CMB as compared to CTRL CX - 5461 - resistant ( CX ) ( representatives of n = 3 ) . ( C ) Quantitation of polysome : monosome in indicated early - passage Eµ - Myc lymphoma as determined by measuring under curve using ImageJ software . Graph represents mean ± of n = 3 .

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(B) Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX-5461-resistant (CX) cells (representatives of n=3). (C) Quantitation of polysome:monosome ratio in the indicated early-passage Eµ-Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software. Graph represents mean ± SEM of n=3.


( F ) Polysomal abundance of mRNAs ( selected on a nominal p - value < 0 . 01 cut - off in either CMB vs . CTRL or CX vs . CTRL as analysed by limma ) encoding components of mitochondrial oxidative phosphorylation on polysome profiling data . Red denotes upregulation , blue denotes downregulation ( n = 3 ; genes with an adjusted P value ( false discovery rate ; FDR ) ≤ 0 . 05 were considered to be significant , are denoted with a black border ) .

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(F) Polysomal abundance of mRNAs (selected based on a nominal p-value < 0.01 cut-off in either the CMB vs. CTRL or CX vs. CTRL as analysed by limma) encoding components of mitochondrial oxidative phosphorylation based on polysome profiling data. Red denotes upregulation, blue denotes downregulation (n=3; genes with an adjusted P value (false discovery rate; FDR) ≤ 0.05 were considered to be significant, and are denoted with a black border).


( A ) Western demonstrating effects of metformin treatment for 48 hours on of active GTP - bound RAP1 in CX CMB ( n = 3 ) its quantitation .

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(A) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP-bound RAP1 in CX and CMB cells (n=3) and its quantitation.


( B , C ) Proportion of green ( GFP ) - positive CMB ( clone # 8 ) in ( B ) lymph node ( C ) spleen of transplanted C57BL / 6 mice treated as indicated for 6 hours on day 12 post - transplant .

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(B, C) Proportion of green fluorescent protein (GFP)-positive CMB (clone #8) cells in (B) lymph node and (C) spleen of transplanted C57BL/6 mice treated as indicated for 6 hours on day 12 post-transplant.


( D ) Kaplan - Meier curve of C57BL / 6 mice transplanted with CX - 5461 - everolimus - resistant ( CMB # 8 ) early - passage Eµ - Myc lymphoma treated with vehicles ( everolimus vehicle : 1 % methylcellulose ; CX - 5461 / metformin vehicle : 25 mM NaH2PO 4 ) ( n = 6 ) ; CX - 5461 ( 35 mg / kg every twice weekly ) everolimus ( 5 mg / kg daily ) ( n = 8 ) , metformin ( 400 mg / kg twice daily ) ( n = 6 ) , or CX - 5461 , everolimus metformin ( 35 mg / kg twice weekly , 5 mg / kg daily 400 mg / kg twice daily , respectively ) ( n = 8 ) . grey : 5 - day metformin pre - treatment period , grey : treatment period . Data were analyzed by a log - rank ( Mantel - Cox ) . Vehicle vs . CX - 5461 - everolimus : P = 0 . 0006 , Vehicle vs . CX - 5461 - everolimus - metformin : P = 0 . 0001 . CX - 5461 - everolimus vs . CX - 5461 - everolimus - metformin : P = 0 . 0003 .

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(D) Kaplan-Meier curve of C57BL/6 mice transplanted with CX-5461-everolimus-resistant (CMB #8) early-passage Eµ-Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX-5461/metformin vehicle: 25 mM NaH2PO4) (n=6); CX-5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily) (n=8), metformin (400 mg/kg twice daily) (n=6), or CX-5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively) (n=8). Light grey: 5-day metformin pre-treatment period, dark grey: treatment period. Data were analyzed by a log-rank (Mantel-Cox) test. Vehicle vs. CX-5461-everolimus: P = 0.0006, Vehicle vs. CX-5461-everolimus-metformin: P = 0.0001. CX-5461-everolimus vs. CX-5461-everolimus-metformin: P = 0.0003.


a - b , Representative of cilia ( a ) dot plots ( b ) showing cilia variation at different time points after serum starvation ( SS ) . Markers in ( a ) show ARL13B ( cilia ) PCNT ( Basal ) . graph shows of cilia from 3 biological replicates ( n > 150 cilia per sample experiment ) . Scale bar : 1µm . Data shown in include mean ± s . d . are calculated by two - unpaired student t - .

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a-b, Representative images of cilia (a) and dot plots (b) showing primary cilia length variation at different time points after serum starvation (SS). Markers in (a) show ARL13B (cilia) and PCNT (Basal body). The graph shows individual length of cilia from 3 biological replicates (n>150 cilia per sample and experiment). Scale bar: 1µm. Data shown in include mean ±s.d. are calculated by two-tailed unpaired student t-test.


d , Representative show cilia segmentation on acetylated tubulin ( Ac tub ) , polyglutamylated tubulin ( Glu tub ) , ARL13B PCNT . cartoon on left indicates proximal segments ( PS , Glu tub rich - region ) distal segments ( DS ) . DS were determined by calculating difference between PS total cilia on ARL13B as depicted by dashed lines on . Scale bar : 1 . 5µm . e , of d . Comparative of average cilia on Ac tubulin ARL13B ( cilia ) shown for specified cilia groups ( 2 < , 2 - 3 , 3 - 4 > 4µm ) . Three biological replicates , n = 400 cilia per sample repetition . numbers above cartoons show of calculated Ac tub / whole cilia . Data shown include mean ± s . d . are calculated by two - unpaired student t - .

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d, Representative images show cilia segmentation based on acetylated tubulin (Ac tub), polyglutamylated tubulin (Glu tub), ARL13B and PCNT. The cartoon on the left indicates proximal segments (PS, Glu tub rich-region) and distal segments (DS). DS were determined by calculating the difference between PS and total cilia length based on ARL13B staining as depicted by the dashed lines on the images. Scale bar: 1.5µm. e, Quantification of d. Comparative analysis of average cilia length based on Ac tubulin and ARL13B (cilia) staining shown for specified cilia length groups (2<, 2-3, 3-4 and >4µm). Three biological replicates, n=400 cilia per sample and repetition. The numbers above the cartoons show the ratio of the calculated Ac tub/whole cilia length. Data shown include mean ±s.d. are calculated by two-tailed unpaired student t-test.


a , Representative of 48h serum starved RPE1 treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) / or nocodazole ( Noco ) for 3h before immunostaining with indicated antibodies . Scale bar : 3µm . b , of ( a ) showing average of proximal segment ( PS , Glu tub ) whole cilia ( ARL13B ) . numbers above cartoons show of calculated distal segment ( DS ) / whole cilia . Three biological replicates , n = 100 cilia per sample repetition . Data shown include mean ± s . d . ; P values are calculated by two - unpaired student t - .

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a, Representative images of 48h serum starved RPE1 cells treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Scale bar: 3µm. b, Quantification of (a) showing average length of proximal segment (PS, Glu tub) and whole cilia (ARL13B). The numbers above the cartoons show the ratio of the calculated distal segment (DS)/whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.


RPE1 were treated with indicated siRNAs before serum starvation for 48h . Representative immunostained with indicated antibodies Scale bar : 2µm .

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RPE1 cells were treated with the indicated siRNAs before serum starvation for 48h. Representative immunostained images with indicated antibodies Scale bar: 2µm.


e - f , were treated with indicated siRNAs before serum starvation for 48h treatment with DMSO or Noco for 3h before immunostaining with indicated antibodies . Representative ( e ) of cilia on ARL13B ( f ) are shown . Scale bar : 2µm . Three biological replicates , n = 100 cilia per sample repetition . Data shown include mean ± s . d . ; P values are calculated by two - unpaired student t - .

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e-f, Cells were treated with the indicated siRNAs before serum starvation for 48h and treatment with DMSO or Noco for 3h before immunostaining with the indicated antibodies. Representative images (e) and quantification of cilia length based on ARL13B (f) are shown. Scale bar: 2µm. Three biological replicates, n=100 cilia per sample and repetition. Data shown include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test.


a , show localisation of endogeneous SEPT2 SEPT7 in RPE1 ARLB13B - mRuby2 with basal . insets to right show enlargements of cilium . Scale bar : 4µm ( large ) 2µm ( small ) . b , STED microscopy showing ciliated RPE1 expressing EGFP - SEPT2 or SEPT7 - EGFP stained for ARL13B . Scale bar : 1µm .

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a, Images show the localisation of endogeneous SEPT2 and SEPT7 in RPE1 ARLB13B-mRuby2 cells with basal body staining. The insets to the right show enlargements of the cilium. Scale bar: 4µm (large) and 2µm (small). b, STED microscopy images showing ciliated RPE1 cells expressing EGFP-SEPT2 or SEPT7-EGFP and stained for ARL13B. Scale bar: 1µm.


d , Representative showing co - localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B - mRuby2 GFP - SEPT2 . arrows point to specific MKS3 or CEP290 signals . Scale bar : 1µm . e , scan measurements show normalised signal intensities along cilium for indicated as specified in ( d ) ( dashed lines ) .

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d, Representative images showing co-localisation of MKS3 or CEP290 in ciliated RPE1 ARLB13B-mRuby2 and GFP-SEPT2 cells. The white arrows point to the specific MKS3 or CEP290 signals. Scale bar: 1µm. e, Line scan measurements show the normalised signal intensities along the cilium for the indicated proteins as specified in (d) (dashed lines).


f , Effect of nocodazole ( Noco ) treatment ( 3h ) on localisation of endogenous SEPT2 SEPT7 at cilia in RPE1 . insets show magnifications of cilium . Scale bar : 5µm ( large ) 2 . 5µm ( small ) . g , Relative signal of endogenous SEPT2 or SEPT7 along cilium from ( f ) . SEPT2 or SEPT7 signals at cilia were normalized to total cilia . Three biological replicates , n > 90 cilia per sample repetition . Data show in ( g ) includes mean ± s . d . ; P values are calculated by two - unpaired student t - . See source data .

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f, Effect of nocodazole (Noco) treatment (3h) on the localisation of endogenous SEPT2 and SEPT7 at cilia in RPE1 cells. The insets show magnifications of the cilium. Scale bar: 5µm (large) and 2.5µm (small). g, Relative signal intensity of endogenous SEPT2 or SEPT7 along the cilium from (f). SEPT2 or SEPT7 signals at cilia were normalized to the total cilia length. Three biological replicates, n>90 cilia per sample and repetition. Data show in (g) includes mean ±s.d.; P values are calculated by two-tailed unpaired student t-test. See source data.


g , Electron microscopy of RPE1 WT SEPT2 - KO cilia . Black arrows point to axoneme basal ( BB ) . Scale bar : 400nm . h , of ( g ) . Only cilia detected from bottom ( BB ) to tip in serial sections were quantified . N > 15 cilia from two independent experiments . Data include mean ± s . d . ; P values are calculated by unpaired Wilcoxon - Mann - Whitney Rank Sum

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g, Electron microscopy of RPE1 WT and SEPT2-KO cilia. Black arrows point to the axoneme and basal body (BB). Scale bar: 400nm. h, Quantification of (g). Only cilia detected from bottom (BB) to tip in serial sections were quantified. N>15 cilia from two independent experiments. Data include mean ±s.d.; P values are calculated by unpaired Wilcoxon-Mann-Whitney Rank Sum Test


i - j , RPE1 WT SEPT2 - KO after 48h SS were treated with solvent control ( DMSO ) , cytochalasin D ( CytoD ) / or nocodazole ( Noco ) for 3h before immunostaining with indicated antibodies . Representative ( i ) quantifications of PS ( Glu tub ) whole cilia ( ARL13B ) ( j ) are shown . numbers above cartoons in ( j ) show of calculated DS / whole cilia . Scale bar : 3µm . Three biological replicates , n = 100 cilia per sample repetition . Data show include mean ± s . d . ; P values are calculated by two - unpaired student t -

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i-j, RPE1 WT and SEPT2-KO cells after 48h SS were treated with solvent control (DMSO), cytochalasin D (CytoD) and/or nocodazole (Noco) for 3h before immunostaining with the indicated antibodies. Representative images (i) and quantifications of PS (Glu tub) and whole cilia (ARL13B) length (j) are shown. The numbers above the cartoons in (j) show the ratio of the calculated DS/whole cilia length. Scale bar: 3µm. Three biological replicates, n=100 cilia per sample and repetition. Data show include mean ±s.d.; P values are calculated by two-tailed unpaired student t-test


of relative signal intensities for indicated ( b ) . IFT88 KIF17 signal intensities were normalized to whole cilia . KIF7 signal was quantified at ciliary tip or base Three biological replicates , n > 80 cilia per sample repetition . Data include mean ± s . d . P values are calculated by two - unpaired student t - .

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quantification of the relative signal intensities for the indicated proteins (b). IFT88 and KIF17 signal intensities were normalized to the whole cilia length. KIF7 signal intensity was quantified at ciliary tip or base Three biological replicates, n>80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


c - d , KIF7 localisation at tip base of cilia ( indicated by arrows ) in RPE1 after 48h SS treated for 3h with solvent control ( DMSO ) or nocodazole ( Noco ) of relative signal intensities for ciliary base tip of KIF7 ( d ) . Scale bar : 2µm . KIF7 signal was quantified at ciliary tip or base as indicated in ( c ) ( arrows ) . Three biological replicates , at least 80 cilia per sample repetition . Data include mean ± s . d . P values are calculated by two - unpaired student t - .

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c-d, KIF7 localisation at the tip and base of cilia (indicated by arrows) in RPE1 cells after 48h SS treated for 3h with solvent control (DMSO) or nocodazole (Noco) and quantification of the relative signal intensities for the ciliary base and tip of KIF7 (d). Scale bar: 2µm. KIF7 signal intensity was quantified at the ciliary tip or base as indicated in (c) (white arrows). Three biological replicates, at least 80 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


e - f , RPE1 WT SEPT2 - KO were treated with control or KIF7 siRNA after 48h SS immunostained with indicated antibodies . Representative ( e ) quantifications of proximal segment ( Glu tub ) whole cilia ( ARL13B ) ( f ) are shown . numbers above cartoons in ( f ) show of calculated distal segments / whole cilia . Three biological replicates , n = 100 cilia per sample repetition . Scale bar : 2µm . Data include mean ± s . d . P values are calculated by two - unpaired student t - .

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e-f, RPE1 WT and SEPT2-KO cells were treated with control or KIF7 siRNA after 48h SS and immunostained with the indicated antibodies. Representative images (e) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (f) are shown. The numbers above the cartoons in (f) show the ratio of the calculated distal segments /whole cilia length. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


d , FRAP experiment of SSTR3 - GFP in RPE1 WT SEPT2 KO . were starved for 48h before beginning of inspection imaged every 30sec for 10mins after bleaching . Scale bar : 5µm . e , of normalised SSTR3 - GFP signal recovery at whole cilia after photobleching from ( d ) . n = 20 cilia are quantified from three biological replicates Data include mean ± s . d . P values are calculated by two - unpaired student t - .

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d, FRAP experiment of SSTR3-GFP in RPE1 WT and SEPT2 KO cells. Cells were starved for 48h before the beginning of inspection and imaged every 30sec for 10mins after bleaching. Scale bar: 5µm. e, Percentage of normalised SSTR3-GFP signal recovery at whole cilia after photobleching from (d). n=20 cilia are quantified from three biological replicates Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


f - g , Influence of MKS3 depletion on cilia of RPE WT SEPT2 - KO . are treated with indicated siRNA serum starved ( SS ) for 48h before immunostaining with indicated antibodies . Representative ( f ) quantifications of proximal segment ( Glu tub ) whole cilia ( ARL13B ) ( g ) are shown . numbers above cartoons in ( g ) show of calculated distal segments / whole cilia . Scale bar : 2µm . Three 3 biological replicates , n = 100 cilia per sample repetition . Data include mean ± s . d . P values are calculated by two - unpaired student t - .

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f-g, Influence of MKS3 depletion on cilia length of RPE WT and SEPT2-KO cells. Cells are treated with the indicated siRNA and serum starved (SS) for 48h before immunostaining with the indicated antibodies. Representative images (f) and quantifications of proximal segment (Glu tub) and whole cilia (ARL13B) length (g) are shown. The numbers above the cartoons in (g) show the ratio of the calculated distal segments/whole cilia length. Scale bar: 2µm. Three 3 biological replicates, n=100 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


h - i , A functional TZ is required for KIF7 cilia tip localisation in RPE1 . KIF7 signal intensities are measured in ciliated RPE1 treated with control or MKS3 siRNA 48h SS . Representative ( h ) of KIF7 signal at base tip of cilia ( h ) are shown . Scale bar : 2µm . Three biological replicates , at least 60 cilia per sample repetition . Data include mean ± s . d . P values are calculated by two - unpaired student t - .

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h-i, A functional TZ is required for KIF7 cilia tip localisation in RPE1 cells. KIF7 signal intensities are measured in ciliated RPE1 cells treated with control or MKS3 siRNA and 48h SS. Representative images (h) and quantification of KIF7 signal intensity at the base and tip of cilia (h) are shown. Scale bar: 2µm. Three biological replicates, at least 60 cilia per sample and repetition. Data include mean ±s.d. and P values are calculated by two-tailed unpaired student t-test.


a , Time - lapse of RPE1 WT SEPT2 - KO stably expressing ARL13B - GFP γ - tubulin - mRuby2 . are starved for 32h before beginning of inspection imaged every 15min for 9 . 5h . Scale bar : 5µm . b , of ( a ) showing changes in cilia during time - lapse imaging . Three biological replicates , 10 cilia per sample repetition . c , Time - lapse show ectocytosis events observed in RPE1 SEPT2 - KO . Experiments are done as in ( a ) with exception that are imaged every 15min . Scale bar : 2 . 5µm .

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a, Time-lapse images of RPE1 WT and SEPT2-KO cells stably expressing ARL13B-GFP and γ-tubulin-mRuby2. Cells are starved for 32h before the beginning of inspection and imaged every 15min for 9.5h. Scale bar: 5µm. b, Quantification of (a) showing changes in cilia length during time-lapse imaging. Three biological replicates, 10 cilia per sample and repetition. c, Time-lapse images show ectocytosis events observed in RPE1 SEPT2-KO cells. Experiments are done as in (a) with exception that cells are imaged every 15min. Scale bar: 2.5µm.


g - h , RPE1 SEPT2 - KO expressing ARL13B - mRuby2 Neongreen - EFHC1 after 48h SS immunostained with Glu - tub antibody . Representative ( g ) quantifications of proximal segment ( Glu tub ) , EFHC1 whole cilia ( ARL13B ) ( h ) are shown . Three biological replicates , n = 100 cilia per sample repetition . Scale bar : 2µm .

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g-h, RPE1 SEPT2-KO cells expressing ARL13B-mRuby2 and Neongreen-EFHC1 after 48h SS and immunostained with Glu-tub antibody. Representative images (g) and quantifications of proximal segment (Glu tub), EFHC1 and whole cilia (ARL13B) length (h) are shown. Three biological replicates, n=100 cilia per sample and repetition. Scale bar: 2µm.


mice , housed at 30 ° C or 6 ° C for 10 days , were intraperitoneally ( IP ) administered with [ U - 13C ] glucose ( 2 g / kg ) . 15 minutes after injection , BAT was harvested for metabolic enrichment . ( A ) m + 6 glucose enrichment in serum BAT . ( B ) relative glucose abundance in serum BAT .

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Male mice, housed at 30°C or 6°C for 10 days, were intraperitoneally (IP) administered with [U-13C]glucose (2 g/kg). 15 minutes after injection, BAT was harvested for metabolic enrichment assay. (A) m+6 glucose enrichment in serum and BAT. (B) relative glucose abundance in serum and BAT.


( D , E ) Metabolic 13C enrichments in BAT of mice are shown as m + 3 glycolytic intermediates ( D ) , m + 2 TCA cycle intermediates ( E ) .

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(D,E) Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates (D), m+2 TCA cycle intermediates (E).


( A ) Representative immunofluorescence shows UCP1 ( green ) , perilipin ( red ) DAPI ( blue ) in sWAT of mice housed at 6 ° C .

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(A) Representative immunofluorescence staining shows UCP1 (green), perilipin (red) and DAPI (blue) in sWAT of mice housed at 6°C.


( B - G ) mice , housed at 30 ° C or 6 ° C for 10 days , were administered with [ U - 13C ] glucose ( 2 g / kg , IP ) . 15 minutes after injection , sWAT gWAT were harvested for metabolic enrichment . Metabolic 13C enrichments in sWAT of mice are shown as m + 6 glucose ( B ) , m + 3 glycolytic intermediates ( C ) , m + 2 TCA cycle intermediates ( D ) . Metabolic 13C enrichments in gWAT of mice are shown as m + 6 glucose ( E ) , m + 3 glycolysis intermediates ( F ) , m + 2 TCA cycle intermediates ( G ) .

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(B-G) Male mice, housed at 30°C or 6°C for 10 days, were administered with [U-13C]glucose (2 g/kg, IP). 15 minutes after injection, sWAT and gWAT were harvested for metabolic enrichment assay. Metabolic 13C enrichments in sWAT of male mice are shown as m+6 glucose (B), m+3 glycolytic intermediates (C), and m+2 TCA cycle intermediates (D). Metabolic 13C enrichments in gWAT of male mice are shown as m+6 glucose (E), m+3 glycolysis intermediates (F), and m+2 TCA cycle intermediates (G).


( H ) Plasma 13CO2 enrichment , after normalizing to [ U - 13C ] glucose tracer enrichment .

(H) Plasma 13CO2 enrichment, after normalizing to the [U-13C]glucose tracer enrichment.


Mouse brown adipocytes were differentiated from freshly isolated in stromal vascular fractions ( SVF ) of interscapular BAT . ( A ) Upper : Oil red of during differentiation . Lower : Relative mRNA of brown adipocyte markers were measured by qPCR .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (A) Upper: Oil red staining of the cells during differentiation. Lower: Relative mRNA levels of brown adipocyte markers were measured by qPCR.


Mouse brown adipocytes were differentiated from freshly isolated in stromal vascular fractions ( SVF ) of interscapular BAT . ( B ) Glycolytic intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (B) Glycolytic intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


Mouse brown adipocytes were differentiated from freshly isolated in stromal vascular fractions ( SVF ) of interscapular BAT . ( C ) TCA cycle intermediates were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours .

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

Mouse brown adipocytes were differentiated from the freshly isolated cells in the stromal vascular fractions (SVF) of the interscapular BAT. (C) TCA cycle intermediates were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose for 2 hours.


( A ) Fully differentiated brown adipocytes were treated with β3 - AR agonist CL316 , 243 ( 10 µM ) . Relative mRNA of thermogenic markers were measured by qPCR .

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(A) Fully differentiated brown adipocytes were treated with β3-AR agonist CL316,243 (10 µM). Relative mRNA levels of thermogenic markers were measured by qPCR.


( B ) Pyruvate lactate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose 10 µM CL316 , 243 for 2 hours .

O O O O O O O O O O O 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) Pyruvate and lactate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( C ) Citrate malate enrichment were analyzed by GC / MS , after cultured with medium containing 10 mM [ U - 13C ] glucose 10 µM CL316 , 243 for 2 hours .

(C) Citrate and malate enrichment were analyzed by GC/MS, after cultured with medium containing 10 mM [U-13C]glucose and 10 µM CL316,243 for 2 hours.


( A ) oxygen consumption rate ( OCR ) of mouse brown adipocytes treated with MPC inhibitor CHC ( 2 mM ) or UK5099 ( 2 µM ) , n = 6 - 7 biological repeats .

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(A) oxygen consumption rate (OCR) of mouse brown adipocytes treated with MPC inhibitor CHC (2 mM) or UK5099 (2 µM), n=6-7 biological repeats.


( B ) Differentiated mouse brown adipocytes were pre - treated with β3 - AR agonist CL316 , 243 for 4 hours , before cultured with medium containing 10 mM [ U - 13C ] glucose for 2 hours with CHC or UK5099 . Metabolic 13C enrichments in brown adipocytes were shown as m + 3 glycolytic intermediates , m + 2 m + 3 TCA cycle intermediates , n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Differentiated mouse brown adipocytes were pre-treated with β3-AR agonist CL316,243 for 4 hours, before cultured with medium containing 10 mM [U-13C]glucose for 2 hours with CHC or UK5099. Metabolic 13C enrichments in brown adipocytes were shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=3 biological replicates.


( C ) After housed at 6 ° C for 10 days , mice were IP injected with PBS or CHC ( 500 mg / kg ) . 30 minutes after CHC treatment , mice were IP administered with [ U - 13C ] glucose ( 2 g / kg ) . Metabolic 13C enrichments in BAT of mice are shown as m + 3 glycolytic intermediates , m + 2 m + 3 TCA cycle intermediates , n = 7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) After housed at 6°C for 10 days, mice were IP injected with PBS or CHC (500 mg/kg). 30 minutes after CHC treatment, mice were IP administered with [U-13C]glucose (2 g/kg). Metabolic 13C enrichments in BAT of male mice are shown as m+3 glycolytic intermediates, m+2 and m+3 TCA cycle intermediates, n=7.


( D ) of CHC ( left : 400 mg / kg , n = 5 ) ( right : 500 mg / kg , n = 7 ) treated mice .

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(D) Body temperature of the CHC (left: 400 mg/kg, n=5) and (right: 500 mg/kg, n=7) treated mice.


B Elution volumes ( peak positions ) of Munc18 - 1 mutants in absence ( top ) presence ( bottom ) of Syx1 using - exclusion chromatography on Superdex - 200 column .

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B Elution volumes (peak positions) of the Munc18-1 mutants in the absence (top) and presence (bottom) of Syx1 using size-exclusion chromatography on Superdex-200 column.


C Effects of domain 3a mutations on MUN - catalyzed transition detected by native - PAGE . Components addition was indicated on top of every lane . D Quantitative of integrated densities of Munc18 - 1 / Syx1 bands in C .

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C Effects of the domain 3a mutations on MUN-catalyzed transition detected by native-PAGE assay. Components addition was indicated on the top of every lane. D Quantitative analysis of the integrated densities of Munc18-1/Syx1 bands in C.


Effects of QKAA , KKEE L348R mutations on MUN - catalyzed transition detected by FRET . Schematic diagram is shown on top of chart . FRET between BODIPY FL - labeled Syb2 ( donor ) TMR - labeled SN25 ( acceptor ) was monitored .

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Effects of the QKAA, KKEE and L348R mutations on MUN-catalyzed transition detected by FRET assay. Schematic diagram is shown on the top of the chart. FRET between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.


F Effects of QKAA , KKEE L348R mutations on lipid mixing between liposomes with Munc18 - 1 / Syx1 liposomes containing Syb2 in presence of Munc13 ( M13 ) , SN25 Syt1 / Ca2 + . Illustration of reactions is shown on top of chart .

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F Effects of the QKAA, KKEE and L348R mutations on lipid mixing between liposomes with Munc18-1/Syx1 and liposomes containing Syb2 in the presence of Munc13 (M13), SN25 and Syt1/Ca2+. Illustration of the reactions is shown on the top of the chart.


A Representative traces ( left ) , summary graphs of normalized frequency ( middle ) normalized amplitude ( right ) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18 - 1 WT or mutants , with or without Munc18 - 1 shRNAs ( Control , n = 25 ; None , n = 17 ; WT , n = 17 ; QKAA , n = 18 ; KKEE , n = 16 ; P335A , n = 23 ; L348R , n = 21 ) . B Sample traces ( left ) , summary graphs of normalized amplitude ( middle ) normalized charge transfer ( right ) of evoked IPSCs recorded from neurons as described in panel A ( Control , n = 19 ; None , n = 19 ; WT , n = 19 ; QKAA , n = 13 ; KKEE , n = 18 ; P335A , n = 17 ; L348R , n = 21 ) . C Sample traces ( left ) of normalized charge transfer evoked by hypertonic sucrose ( right ) recorded from neurons described in panel A ( Control , n = 17 ; None , n = 13 ; WT , n = 17 ; QKAA , n = 17 ; KKEE , n = 18 ; P335A , n = 15 ; L348R , n = 14 ) .

O O O O O O O O O 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 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

A Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21). B Sample traces (left), summary graphs of normalized amplitude (middle) and normalized charge transfer (right) of evoked IPSCs recorded from neurons as described in panel A (Control, n = 19; None, n = 19; WT, n = 19; QKAA, n = 13; KKEE, n = 18; P335A, n = 17; L348R, n = 21). C Sample traces (left) and quantification of the normalized charge transfer evoked by hypertonic sucrose (right) recorded from the neurons described in panel A (Control, n = 17; None, n = 13; WT, n = 17; QKAA, n = 17; KKEE, n = 18; P335A, n = 15; L348R, n = 14).


A Representative traces ( left ) , summary graphs of normalized frequency ( middle ) normalized amplitude ( right ) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18 - 1 WT or mutants , with or without Munc18 - 1 shRNAs ( Control , n = 25 ; None , n = 17 ; WT , n = 17 ; QKAA , n = 18 ; KKEE , n = 16 ; P335A , n = 23 ; L348R , n = 21 ) .

O O O O O O O O O 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 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

A Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21).


A Representative of HEK293T transfected with mCherry2 - Syx1 or EGFP - Munc18 - 1 . Lipophilic dye DiD ( 1 , 1 - Dioctadecyl - 3 , 3 , 3 , 3 - tetramethylindodicarbocyanine ) was used as plasma marker . B Representative of HEK293T co - transfected with mCherry2 - Syx1 EGFP labeled WT or mutants of Munc18 - 1 .

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A Representative images of HEK293T cells transfected with mCherry2-Syx1 or EGFP-Munc18-1. Lipophilic dye DiD (1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) was used as the plasma membrane marker. B Representative images of HEK293T cells co-transfected with mCherry2-Syx1 and EGFP labeled WT or mutants of Munc18-1.


C , D Summary graphs of distribution of Syx1 ( C ) Munc18 - 1 WT or mutants ( D ) quantified by of at plasma ( PM ) to total in whole ( total ) . DiD is PM marker . Numbers of pictures analyzed are shown as follows : no Syx1 , n = 14 ; no M18 , n = 15 ; WT , n = 24 ; QKAA , n = 23 ; KKEE , n = 12 ; L348R , n = 22 .

O O O O O O O O B-MEASURED_VAR 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 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

C,D Summary graphs of the membrane distribution of Syx1 (C) and Munc18-1 WT or mutants (D) quantified by the ratio of fluorescence at the plasma membrane (PM) to the total fluorescence in the whole cell (total). DiD is the PM marker. Numbers of pictures analyzed are shown as follows: no Syx1, n = 14; no M18, n = 15; WT, n = 24; QKAA, n = 23; KKEE, n = 12; L348R, n = 22.


E Munc18 - 1 mutants retain interaction with Syx1 shown by GST pull - down experiment . A representative gel displayed is from one of three independent replicates .

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E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.


B Example histograms of smFRET efficiency of Syx1 in with Munc18 - 1 WT in absence presence of MUN domain , within SNARE . C − E Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 QKAA ( C ) , KKEE ( D ) , L348R ( E ) in absence or presence of MUN domain . QKAA mutation strongly impaired shift of smFRET efficiency in presence of MUN domain .

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B Example histograms of smFRET efficiency of Syx1 in complex with Munc18-1 WT in the absence and presence of the MUN domain, and within the SNARE complex. C−E Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 QKAA (C), KKEE (D), and L348R (E) in the absence or presence of the MUN domain. The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain.


F Quantitative results of Gaussian peak center positions of FRET efficiency distributions in C − E .

F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C−E.


G Effects of QKAA , KKEE , L348R mutations on interaction between Munc18 - 1 / Syx1 MUN domain by GST pull - down combined with immunoblotting . Results were shown with 6 × His - tag antibody GST - tag antibody .

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G Effects of QKAA, KKEE, and L348R mutations on the interaction between the Munc18-1/Syx1 complex and the MUN domain by GST pull-down assay combined with immunoblotting. Results were shown with 6×His-tag antibody and GST-tag antibody.


B Effects of QKAA , KKEE , L348R mutations on binding of Syb2 to Munc18 - 1 / Syx1 in presence absence of MUN domain detected by GST pull - down . Bands of Munc18 - 1 are indicated with red asterisks . C of normalized binding of Syb2 to Munc18 - 1 / Syx1 . Integrated intensities of Munc18 - 1 bands were quantified . QKAA L348R , but not KKEE , disrupted Syb2 binding to Munc18 - 1 / Syx1 .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_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 B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O

B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks. C Quantification of the normalized binding of Syb2 to Munc18-1/Syx1 complex. Integrated intensities of Munc18-1 bands were quantified. QKAA and L348R, but not KKEE, disrupted Syb2 binding to the Munc18-1/Syx1 complex.


B Effects of QKAA , KKEE , L348R mutations on binding of Syb2 to Munc18 - 1 / Syx1 in presence absence of MUN domain detected by GST pull - down . Bands of Munc18 - 1 are indicated with red asterisks .

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

B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks.


A Ensemble FRET showing different effects of Munc18 - 1 P335A mutation Syx1 LEAA mutation in transition from Munc18 - 1 / Syx1 to SNARE with without MUN domain . FRET signal between BODIPY FL - labeled Syb2 ( donor ) TMR - labeled SN25 ( acceptor ) was monitored . B of FRET results in A .

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A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored. B Quantification of the FRET results in A.


A Ensemble FRET showing different effects of Munc18 - 1 P335A mutation Syx1 LEAA mutation in transition from Munc18 - 1 / Syx1 to SNARE with without MUN domain . FRET signal between BODIPY FL - labeled Syb2 ( donor ) TMR - labeled SN25 ( acceptor ) was monitored .

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

A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.


C Single - molecule FRET showing MUN domain drives open of Syx1 linker region independent of conformational change of domain 3a . Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 P335A mutant in absence presence of MUN domain . D Quantitative results of Gaussian peak center positions of FRET efficiency distributions in C .

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

C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain. D Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C.


C Single - molecule FRET showing MUN domain drives open of Syx1 linker region independent of conformational change of domain 3a . Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 P335A mutant in absence presence of MUN domain .

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

C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain.


B Munc18 - 1 , Habc H3 construct a tertiary .

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B Munc18-1, Habc and H3 construct a tertiary complex.


C tertiary globally resembles Munc18 - 1 / Syx1 .

C The tertiary complex globally resembles the Munc18-1/Syx1 complex.


D Formation of tertiary with Munc18 - 1 QKAA , KKEE , L348R mutants . KKEE strongly impaired formation of tertiary .

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D Formation of the tertiary complex with Munc18-1 QKAA, KKEE, and L348R mutants. KKEE strongly impaired formation of the tertiary complex.


E Munc18 - 1 displays interaction with Syx1 - Habc .

E Munc18-1 displays interaction with Syx1-Habc.


F QKAA , KKEE , L348R retained interaction with Syx1 - Habc .

F QKAA, KKEE, and L348R all retained interaction with Syx1-Habc.