( B ) Immunocytochemistry for lamp2a ( left ) , lamp1 ( center ) or lamp2s ( right ) in hematoxylin eosin ‐ stained sections from same livers . Bar : 50 μm . Black arrows : hepatocytes ; arrows : Kupffer .

O O O O O B-MEASURED_VAR 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) Immunocytochemistry for lamp2a (left), lamp1 (center) or lamp2s (right) in hematoxylin and eosin‐stained sections from the same livers. Bar: 50 μm. Black arrows: hepatocytes; white arrows: Kupffer cells.


( C ) Immunofluorescence for lamp2a ( top ) P ‐ glycoprotein ( bottom ) in mouse liver sections . right panel shows merged . Bar : 100 μm . Inset shows detailed at higher magnification . Bar : 50 μm .

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

(C) Immunofluorescence for lamp2a (top) and P‐glycoprotein (bottom) in mouse liver sections. The right panel shows merged images. Bar: 100 μm. Inset shows detailed area at higher magnification. Bar: 50 μm.


( A ) Autofluorography of sequential immunoprecipitation of lamp2a first ( left ) remaining isoforms of lamp2 second ( right ) from metabolically labeled wild ‐ type PPCA ( − / − ) mouse skin fibroblasts . Values are means ± SE of three different experiments similar to ones shown in upper panels .

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

(A) Autofluorography of the sequential immunoprecipitation of lamp2a first (left) and all the remaining isoforms of lamp2 second (right) from metabolically labeled wild‐type and PPCA(−/−) mouse skin fibroblasts. Values are means ± SE of three different experiments similar to the ones shown in the upper panels.


( B ) Immunoblot for lamp2a ( top ) lamp2 isoforms ( bottom ) in lysosomalmembranes from wild ‐ type ( WT ) or PPCA ( − / − ) mouseskinfibroblasts at different times after incubation at 37 ° C . open arrowhead indicates truncated form of lamp2 lacking / transmembrane region ( Cuervo Dice , 2000b ) . Right shows densitometric ( mean ± SE ) of four different experiments similar to one shown here . Values are expressed as of remaining lamp2a , are means ± 1 SE of three different experiments .

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

(B) Immunoblot analysis for lamp2a (top) and all lamp2 isoforms (bottom) in lysosomalmembranes from wild‐type (WT) or PPCA(−/−) mouseskinfibroblasts at different times after incubation at 37°C. The open arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). Right shows the densitometric quantification (mean ± SE) of four different experiments similar to the one shown here. Values are expressed as percentage of remaining lamp2a, and are means ± 1 SE of three different experiments.


( A ) Rates of total degradation in presence absence of serum of normal skin fibroblasts ( CTR ) , GS ( None ) GS supplemented with precursor forms of native PPCA ( + PPCA ) , a catalytic mutant of PPCA ( + PPCA S / A ) or cathepsin L ( Cath L ) . Values are means ± SE of six different experiments . Differences from control value were significant for P 0 . 001 ( * * ) P 0 . 05 ( * ) .

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

(A) Rates of total protein degradation in the presence and absence of serum of normal skin fibroblasts (CTR), GS cells (None) and GS cells supplemented with the precursor forms of native PPCA (+PPCA), a catalytic mutant of PPCA (+PPCA S/A) or cathepsin L (Cath L). Values are means ± SE of six different experiments. Differences from the control value were significant for P 0.001 (**) and P 0.05 (*).


( B ) Immunoblot for lamp2a at time 0 or after 40 min incubation at 37 ° C of lysosomes isolated from PPCA ( − / − ) ( None ) PPCA ( − / − ) supplemented as in ( A ) . At 40 min immunoblot with antibody against luminal region of lamp2a is depicted to show truncated form of lamp2a ( open arrowhead ) . Right : densitometric for lamp2a ( means + range of values ) of two different experiments similar to one shown . Values are expressed as of lamp2a present in non ‐ supplemented .

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

(B) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.


( C ) Immunoblot for lamp2a at time 0 or after 40 min incubation at 37 ° C of lysosomes isolated from PPCA ( − / − ) ( None ) PPCA ( − / − ) supplemented as in ( A ) . At 40 min immunoblot with antibody against luminal region of lamp2a is depicted to show truncated form of lamp2a ( open arrowhead ) . Right : densitometric for lamp2a ( means + range of values ) of two different experiments similar to one shown . Values are expressed as of lamp2a present in non ‐ supplemented .

(C) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.


( D ) Immunocytochemistry for lamp2a of liver ( top ) kidney ( bottom ) sections of ‐ matched wild ‐ type ( WT ) , PPCA ( − / − ) bone marrow transplanted ( BMT ) mice . Bar , 50 μm ( liver ) or 100 μm ( kidney ) .

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 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

(D) Immunocytochemistry for lamp2a of liver (top) and kidney (bottom) sections of age‐matched wild‐type (WT), PPCA(−/−) and bone marrow transplanted (BMT) mice. Bar, 50 μm (liver) or 100 μm (kidney).


( A ) Left : acid sequences of C ‐ terminal regions of lamp2a from different species of other lamp2 isoforms lamp1 from mouse . proposed cleavage site for PPCA in lamp2a is highlighted . Right : immunoblot for HA of lysosomal membranes isolated from two different clones of HEK293 ( mouse ; upper panel ) NIH 3T3 ( human ; lower panel ) stably transfected with wild ‐ type ( FL ) mutated ( GL ) HA ‐ lamp2a . arrowhead indicates truncated form of lamp2 lacking / transmembrane region .

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

(A) Left: amino acid sequences of the C‐terminal regions of lamp2a from different species and of other lamp2 isoforms and lamp1 from mouse. The proposed cleavage site for PPCA in lamp2a is highlighted. Right: immunoblot analysis for HA of lysosomal membranes isolated from two different clones of HEK293 (mouse; upper panel) and NIH 3T3 cells (human; lower panel) stably transfected with wild‐type (FL) and mutated (GL) HA‐lamp2a. The arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region.


( B ) Total rates of degradation in presence absence of serum in non ‐ transfected HEK293 ( WT ) in transfected HEK293 clones described in ( A ) . Values are expressed as of initially radiolabeled remaining at each time are means ± SE of three different experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Total rates of protein degradation in the presence and absence of serum in non‐transfected HEK293 cells (WT) and in the transfected HEK293 clones described in (A). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of three different experiments.


( C ) Top : immunoblot with an anti ‐ HA antibody of purified wild ‐ type ( FL ) mutated ( GL ) mouse HA ‐ lamp2a incubated in an acidic pH buffer alone , or with PPCA . Bottom : wild ‐ type HA ‐ lamp2a was incubated as in top panel without additions ( None ) or in presence of 2 mM EDTA , 10 μM L ‐ trans ‐ epoxysuccinyl ‐ leucylamide ‐ ( 4 ‐ guanido ) ‐ butane ( E ‐ 64 ) , 2 μM pepstatin A ( Pepst ) or 10 μM ABSF . open arrowhead indicates truncated form of lamp2a .

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

(C) Top: immunoblot analysis with an anti‐HA antibody of purified wild‐type (FL) and mutated (GL) mouse HA‐lamp2a incubated in an acidic pH buffer alone, or with PPCA. Bottom: wild‐type HA‐lamp2a was incubated as in the top panel without additions (None) or in the presence of 2 mM EDTA, 10 μM L‐trans‐epoxysuccinyl‐leucylamide‐(4‐guanido)‐butane (E‐64), 2 μM pepstatin A (Pepst) or 10 μM ABSF. The open arrowhead indicates the truncated form of lamp2a.


( D ) Electron microscopy from purified 40S 60S ribosomal complexes ( 1 pmol each ) assembled in vitro in presence of MgCl2 , in presence or absence of 5 pmol of ( PR ) 20 . Assembled 80S particles are indicated ( red arrows ) . Scale bar ( ) represents 10 nm . ( E ) of 80S particles identified in ( D ) ( n = 1000 ) in non - assembly ( 1 mM MgCl2 ) or assembly ( 5 mM MgCl2 ) conditions . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron microscopy images from purified 40S and 60S ribosomal complexes (1 pmol each) assembled in vitro in the presence of MgCl2, and in the presence or absence of 5 pmol of (PR)20. Assembled 80S particles are indicated (red arrows). Scale bar (white) represents 10 nm. (E) Quantification of 80S particles identified in (D) (n=1000) in non-assembly (1 mM MgCl2) or assembly (5 mM MgCl2) conditions. (


( C , D ) High - throughput microscopy ( HTM ) - mediated of 5 - ethynyl - uridine ( EU ) ( C ) polyA + mRNA ( D ) per nucleus found in U2OS exposed to 10 μM ( PR ) 20 for 16 h . EU was added 30 ´ prior to fixation ( n = 3 ) . Black lines indicate median values in both panels . In D , boxes define upper lower quartiles whiskers mark highest lowest observations . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - . Representative from these analyses are provided in each case . Scale bar ( ) indicates 2 - 5 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 O O O O O O O O O O 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) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h. EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.


( E ) Effect of ( PR ) 20 ( 10 μM , 16 h ) in number of Cajal Bodies ( CB ) identified by immunofluorescence with anti - COILIN anti - SMN antibodies ( n = 3 ) . Data represent mean values ± SD . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - . Representative from these analyses are provided in each case . Scale bar ( ) indicates 2 - 5 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 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 O O O O O O O O O O O O O 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) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.


( B , C ) of GAPDH or 5 . 8 rRNA quantified by qPCR in reactions containing increasing doses of ( PR ) 20 ( n = 3 ) . Data represent mean values ± . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - .

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

(B, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.


( D ) HTM - mediated of 5 - ethynyl - deoxyuridine ( EdU ) per nucleus found in U2OS exposed to 10 μM ( PR ) 20 for 4 h . EdU was added 30 ´ prior to fixation . Representative from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( ) indicates 5 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

(D) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( E ) HTM - mediated of γH2AX per nucleus in U2OS exposed to 3Gy of IR in presence or absence of 10 μM ( PR ) 20 for indicated times . Note accumulation of with persistent damage ( γH2AX ) 10 h after IR in presence of DPR . Representative from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( ) indicates 5 μm .

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 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

(E) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( D ) Immunofluorescence of Cy3 - labelled salmon protamine ( red ) nucleolar factor UBF1 ( green ) in U2OS treated with 30 μM Cy3 - protamine for 4 h . Scale bar ( ) indicates 2 . 5 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 O O O O O O O O O O O O O O O O O O O O

(D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.


( E , F ) HTM - mediated of EU per nucleus ( E ) O - propargyl - puromycin ( OPP ) per ( F ) in U2OS exposed to 30 μM protamine for 16 ( E ) or 24 ( F ) h . EU OPP were added 30 ´ prior to fixation . Black lines indicate median values ( * * * * , p < 0 . 0001 ; t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).


( D ) Immunofluorescence of HA - ( PR ) 20 ( green ) nucleolar factor UBF1 ( red ) in U2OS treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h . Data information : Scale bar ( ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( E ) Immunofluorescence of HA - ( PR ) 20 ( green ) Cy5 ( red ) in U2OS treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 4 μM of Cy5 - labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h . Data information : Scale bar ( ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( F ) Clonogenic survival of U2OS exposed to 7 . 5 μM with indicated peptides or protamine ( PROT . ) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0 . 5 μM Heparin .

O O O O O O O O O O O O O O O O O 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) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.


( G ) Immunofluorescence of HA - ( PR ) 20 ( green ) nucleolar factor UBF1 ( red ) in U2OS treated with 7 . 5 μM HA - ( PR ) 20 alone or together with heparin ( 0 . 5 μM ) . Data information : Scale bar ( ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - .

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 O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( A ) Immunofluorescence of neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 48 hours , respectively , with or without 2 μM of a non - coding 38nt oligonucleotide . Nuclei were stained with DAPI ( blue ) . Scale bar ( ) , 10 μm . Note that besides its impact on overall survival , presence of oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by peptide . ( B , C ) viability quantified CellTiter - Glo luminescence assays mMN ( B ) NSC34DIFF ( C ) treated as in ( A ) ( n = 3 ) . Data represent mean values ± . ( Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide. (B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM. ( Data information: **, p<0.01; ***, p<0.001.


( D ) Immunofluorescence of neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 48 hours , respectively , with or without heparin ( 0 . 5 μM ) . Nuclei were stained with DAPI ( blue ) . Scale bar ( ) , 10 μm . ( E , F ) viability quantified CellTiter - Glo luminescence assays mMN ( E ) NSC34DIFF ( F ) treated as in ( D ) ( n = 3 ) . Data represent mean values ± . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 . S

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. (E, F) Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001. S


of E2F7 E2F8 during cycle progression . HeLa were synchronized by a double thymidine block released into fresh medium . were harvested at indicated time points an asynchronous ( AS ) condition was used as control . were measured by immunoblotting cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . asterisk indicates E2F7 - specific band .

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

Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.


Decreased stability of E2F7 E2F8 in nocodazole - arrested . HeLa were either treated with DMSO or nocodazole ( 50 ng / ml ) for 16 hours . were harvested lysed for immunoblotting . expression of cyclin B1 was used as a marker for G2 or M , γ - tubulin was used as loading control .

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 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

Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.


Selective Cullin - RING inhibitor MLN4924 rescued degradation of E2F7 / 8 under nocodazole - arrested condition . HeLa were treated with DMSO , nocodazole or nocodazole plus MLN4924 ( 0 . 1 μM ) for 16 hours . were harvested lysed for immunoblotting . cyclin B1 expression was used as a marker for G2 or M cycle progression , γ - tubulin was used as loading control .

O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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

Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.


Increased half - life of E2F7 / 8 by MLN4924 treatment . HeLa were either treated with cycloheximide ( CHX , 50 ug / ml ) with or without MLN4924 ( 0 . 1 μM ) . of E2F7 E2F8 were determined by immunoblotting ( left panel ) . Asterisk indicates E2F7 - specific band . cyclin B1 cyclin A2 expressions were used as a marker for G2 or M cycle progression , γ - tubulin was used as loading control . Quantifications ( right panels ) were performed on two independent experiments . Bar error bars represent mean ± .

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 B-MEASURED_VAR I-MEASURED_VAR O 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 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

Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.


C - terminus motifs at parallel positions on E2F7 E2F8 are essential for binding to cyclin F . residues on each motif were mutated to alanines ( R to A , I / L to A ) with site - direct mutagenesis PCR . HEK293 were transfected with either EGFP - tagged empty vector ( EGFP ) , wild - types E2F7 / 8 ( WT ) or alanine mutants . Nocodazole ( 50 ng / ml ) was added 32 hours after transfection , MG132 ( 1 μg / ml ) was added 5 hours before harvesting at 48 hours post transfection . were harvested lysed for immunoprecipitation using anti - EGFP resin followed by immunoblotting with antibodies against cyclin F EGFP .

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

C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.


Schematic view showing truncated mutants ML / AA mutant of cyclin F ( Left ) . HEK were transfected with indicated constructs co - immunoprecipitation was performed using Flag resin ( Right ) .

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

Schematic view showing the truncated mutants and ML/AA mutant of cyclin F (Left). HEK cells were transfected with indicated constructs and co-immunoprecipitation was performed using Flag resin (Right).


Wild - type or mutant versions of EGFP - tagged E2F7 / 8 were co - transfected with either empty vector or Flag - tagged cyclin F in HEK293 . Nocodazole was added to 8h before harvest . 48 hours after transfection , were collected lysed for immunoblotting .

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 B-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

Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.


Knockdown of cyclin F stabilized E2F7 E2F8 . HeLa RPE were transfected with either scramble siRNA or pool cyclin F siRNA . were harvested at 48 hours post transfection . of E2F7 / 8 were analyzed by immunoblotting . Asterisk indicates specific band of E2F7 detection .

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

Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection.


Cyclin F targets atypical E2Fs during G2 / M . HeLa were transfected with either scrambled siRNA ( scr ) or cyclin F siRNA ( sicyclin F ) for 24 hours . Then were synchronized by double thymidine block released into fresh medium after second block . were harvested at indicated timepoints after release . expression was measured by immunoblotting cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . Asterisk indicates specific detection of endogenous E2F7 .

B-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 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 B-MEASURED_VAR O

Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.


Cyclin F contributes to ubiquitylation of E2F7 E2F8 in vivo . HEK293 were transfected with HA - E2F7 / 8 , with or without Flag - cyclin F , with HA - tagged ubiquitin . 5 hours before harvest , were treated with MG132 . 48 hours after transfection , HEK were harvested lysed for immunoprecipitation pull - down with anti - HA resin followed by immunoblotting .

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

Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.


Schematic view of experimental setting for HU - synchronized live imaging . 48 hours before imaging , RPE - FUCCI were transfected with siRNA against scramble or cyclin F . 16 hours before imaging , were synchronized at G1 / S border by HU ( 2 mM ) treatment . Representative from different channels are shown , arrows in Differential Interference Contrast DIC channel indicate traced . Scale bar : 10µm .

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

Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.


of number of Ctrl ( left panel ) 7 / 8KO ( right panel ) RPE - FUCCI with scr or sicyclin F that completed mitosis after HU release . For each condition , 100 were monitored by live imaging . Each was followed until it successfully finished mitosis divided into two daughter for a maximum of 24 hours . Log - rank were performed to analyze statistical significance .

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

Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.


Schematic view of experimental setting for live imaging of asynchronous . 48 hours before imaging , RPE - FUCCI were transfected with siRNA against scramble or cyclin F . At start of imaging , G1 ( red : mKO2 - Cdt1 > mAG1 - Geminin , 50 per condition ) were enrolled subsequently monitored though their entire cycle until mitosis . black dot represents mitosis .

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

Schematic view of the experimental setting for live imaging of asynchronous cells. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. At the start of the imaging, G1 cells (red: mKO2-Cdt1 > mAG1-Geminin, 50 cells per condition) were enrolled and subsequently monitored though their entire cell cycle until mitosis. The black dot represents mitosis.


Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 E2F8 . Number of Ctrl ( left panel ) or 7 / 8KO ( right panel ) RPE - FUCCI with scr or cyclin F RNAi that finished mitosis during live imaging is shown . For each condition , 50 at G1 were monitored by live imaging . Each was followed until it successfully progressed through S G2 phase , finished mitosis divided into two daughter , for a maximum of 24 hours . Log - rank were performed to analyze statistical significance .

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

Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.


Loss of cyclin F delays progression from G1 / S transition to mitosis . Histogram shows time from G1 / S ( mAG1 - Geminin increases to higher than 10 % of maximum value in three consecutive imaging frames ) to completed mitosis . Only that finished mitosis were enrolled in this . Student t - was used to statistical significance , asterisks indicates P value < 0 . 01 . Bar error bars represent mean ± .

O O B-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-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

Loss of cyclin F delays the progression from G1/S transition to mitosis. Histogram shows the time from G1/S (mAG1-Geminin intensity increases to higher than 10% of the maximum value in three consecutive imaging frames) to completed mitosis. Only cells that finished mitosis were enrolled in this quantification. Student t-test was used to test the statistical significance, asterisks indicates the P value <0.01. Bar and error bars represent mean ± SEM.


Depletion of cyclin F stalls cycle at late S / G2 . After 24 hours of live imaging , cycle progression from panel E was quantified . Histogram shows of at each stage ( at 24h ) over whole population ( 50 per condition ) .

O O B-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

Depletion of cyclin F stalls the cell cycle at late S/G2. After 24 hours of live cell imaging, the cell cycle progression from panel E was quantified. Histogram shows the percentage of cells at each stage (at 24h) over the whole population (50 cells per condition).


Disruption of cyclin F binding site increased stability of E2F7 . E2F7WT E2F7R894A constructs were integrated in to HeLa / TO system . were arrested with HU for 16 hours before releasing into doxycycline containing medium , were collected every 3 hours for immunoblotting .

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

Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.


qPCR showing that mRNA level of E2F7WT E2F7R894A were at comparable . Bar error bars represent mean ± .

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

qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM.


Over - expression of E2F7R894A delays cycle progression through G2 - M phase . HeLa / TO expressing either wild - type or mutant version of E2F7 were arrested with 16h of HU , then were released into fresh medium with or without doxycycline . Live imaging was performed to trace G2 - M progression of HeLa / TO .

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 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

Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.


showing differentially expressed genes after cyclin F knockdown , rescued by additional E2F7 / 8 depletion . Highlighted genes are involved in repair . were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cycle progression between different conditions .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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

Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.


qPCR showing expression of atypical E2F - target genes that are involved in replication or repair . HeLa were transfected for 48 hours with siRNAs as indicated . were incubated with nocodazole for 16 hours before harvesting . Data represent averages ± ( n = 3 ) ; * P < 0 . 05 or * * P < 0 . 01 ( Student ' s t - ) . n . s . : not significant .

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

qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.


Immunoblotting showing of Chk1 Rad51 in indicated siRNA conditions . HeLa were treated with nocodazole for 16 hours prior to harvesting .

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

Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.


Loss of cyclin F induced E2F7 / 8 - dependent Homologous Recombination deficiency . HeLa that were stably transformed with pDR - GFP were transfected with siRNA as indicated . 48h after initial transfection , were harvested for Flow - cytometry . GFP positive were gated ( Fig EV5A ) . Relative HR efficiency were adjusted to scramble siRNA condition . Data represent averages ± ( n = 3 ) ; * * P < 0 . 01 ( Student ' s t - ) . n . s . : not significant .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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

Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant.


Loss of cyclin F induced E2F7 / 8 - dependent γ - H2AX accumulation . HeLa were transfected with indicated siRNA for 24 hours , then treated with nocodazole for 16 hours before fixation for immunofluorescence of γ - H2AX . DAPI was used to stain nucleus . Relative of γ - H2AX was quantified by Image J software , 150 were quantified for each condition . Red bars represent averages ; * * P < 0 . 01 ( Student ' s t - ) . n . s . : not significant . Scale bar 20 μm .

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

Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm.


Loss of cyclin F increased damage recovery time before division . RPE integrated with 53BP1 construct were transfected with indicated siRNA for 24 hours , then treated with HU for 16 hours . At beginning of imaging , only single with at least one 53BP1 foci were traced , till time frame that no 53BP1 foci was observed . mitotic progression of was defined as duration from damage recovery to division . Histogram shows damage recovery time ( green ) mitotic progression ( black ) of 50 for each condition . Chi - square was performed to statistical significance ( P < 0 . 01 ) .

O O B-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 B-MEASURED_VAR 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

Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01).


Knockdown of cyclin F caused a delay in lesion recovery that is dependent on E2F7 / 8 . cumulative curves represent add - up number of that overcome damage lesions , for a time frame of 24 hours . For each condition , 50 were quantified . Log - rank were performed to analyze statistical significance .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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

Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7/8. The cumulative curves represent the add-up number of cells that overcome the DNA damage lesions, for a time frame of 24 hours. For each condition, 50 cells were quantified. Log-rank tests were performed to analyze the statistical significance.


Wild - type LGP2 - KO HEK293 in 24 - well plates were transfected with 200 ng / ml of short poly I : C IFN - β IP - 10 mRNA expression was determined by RT - qPCR . data represent mean ± SD ( n = 3 , t - , * p < 0 . 05 ) . LGP2 KO2 KO3 are independently isolated LGP2 KO clones . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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 B-MEASURED_VAR I-MEASURED_VAR I-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 B-CONTROLLED_VAR 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

Wild-type and LGP2-KO HEK293 cells in 24-well plates were transfected with 200 ng/ml of short poly I:C IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). LGP2 KO2 and KO3 are independently isolated LGP2 KO clones. Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


Wild - type LGP2 - KO HEK293 in 24 - well plates were infected with SeV . IFN - β IP - 10 mRNA expression was determined by RT - qPCR . data represent mean ± SD ( n = 3 , t - , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

O O O B-CONTROLLED_VAR 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 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

Wild-type and LGP2-KO HEK293 cells in 24-well plates were infected with SeV. IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( F ) HEK293 in 24 - well plates were transfected with LGP2 ( 0 . 1 , 0 . 3 , or 0 . 6 μg / well ) RIG - I ( 0 . 1 μg / well ) expression vectors as indicated . 24 h after transfection , were stimulated with 200 ng of short poly I : C for 4 hr . Total RNAs were extracted , expression of IFN - β mRNA was determined by RT - qPCR normalized to GAPDH ( n = 3 , t - , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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 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 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

(F) HEK293 cells in 24-well plates were transfected with LGP2 (0.1, 0.3, or 0.6 μg/well) and RIG-I (0.1 μg/well) expression vectors as indicated. 24 h after transfection, cells were stimulated with 200 ng of short poly I:C for 4 hr. Total RNAs were extracted, and the expression of IFN-β mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, t-test, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( G ) HEK293FT were transfected with FLAG - tagged LGP2 HA - tagged ubiquitin expression vectors . Whole extracts ( WCE ) were prepared 24 h after transfection . Immunoprecipitation was performed with anti - FLAG antibody ( IP : FLAG ) were detected by western blot with indicated antibodies .

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 O O O O O O 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) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged ubiquitin expression vectors. Whole cell extracts (WCE) were prepared 24 h after transfection. Immunoprecipitation was performed with anti-FLAG antibody (IP: FLAG) and the proteins were detected by western blot analysis with the indicated antibodies.


( H ) FLAG - tagged LGP2 was transfected into HEK293 . were then infected with SeV at MOI = 10 for 24 h . Immunoprecipitation was performed with anti - FLAG antibody , were detected with indicated antibodies .

O O O O O O O O O O O O O O O O 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) FLAG-tagged LGP2 was transfected into HEK293 cells. Cells were then infected with SeV at MOI = 10 for 24 h. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected with the indicated antibodies.


A ) HEK293 were transfected with FLAG - RIG - I or FLAG - LGP2 expressing vector . were then infected with SeV , whole extracts were collected at indicated time points . Immunoprecipitation was performed with anti - FLAG antibody . were subjected to SDS - PAGE detected by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were transfected with FLAG-RIG-I or FLAG-LGP2 expressing vector. Cells were then infected with SeV, and whole cell extracts were collected at indicated time points. Immunoprecipitation was performed with anti-FLAG antibody. The proteins were subjected to SDS-PAGE and detected by western blotting.


B ) A549 transfected with HA - tagged ubiquitin were stimulated with 200 ng / ml of short poly I : C for 6 h . PLA was performed with anti - HA , anti - RIG - I , anti - LGP2 antibodies . number of PLA signals were counted ( t - , * p < 0 . 05 , n = 20 ) . Each dot represents number of PLA signals of each . Data information : Scale bars represent 10 μm . data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

B) A549 cells transfected with HA-tagged ubiquitin were stimulated with 200 ng/ml of short poly I:C for 6 h. PLA was performed with anti-HA, anti-RIG-I, and anti-LGP2 antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 20). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C ) HEK293 were transfected with empty LGP2 expression vectors for 24 hr . PLA was performed with anti - LGP2 anti - K63 - Ub antibodies . number of PLA signals were counted ( t - , * p < 0 . 05 , n = 33 ) . Each dot represents number of PLA signals of each . Data information : Scale bars represent 10 μm . data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were transfected with empty and LGP2 expression vectors for 24 hr. PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 33). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


D - F ) HEK293 were transfected with 200 ng / ml of short poly I : C . were fixed at indicated time points , PLA was performed with anti - RIG - I , anti - LGP2 , anti - K63 - Ub antibodies ( D ) . number of PLA signals were counted ( E , F ) ( t - , * p < 0 . 05 , n = 30 ) . Each dot represents number of PLA signals of each . Data information : Scale bars represent 10 μm . data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

D-F) HEK293 cells were transfected with 200 ng/ml of short poly I:C. Cells were fixed at the indicated time points, and PLA was performed with anti-RIG-I, anti-LGP2, and anti-K63-Ub antibodies (D). The number of PLA signals were counted (E, F) (t-test, *p < 0.05, n = 30). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


A ) HEK293FT were transfected with FLAG - tagged LGP2 HA - tagged Riplet expression vectors . 24 h after transfection , were lysed , WCE were prepared . Immunoprecipitation was performed with anti - FLAG antibody , were detected by western blotting with indicated antibodies .

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 O O 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) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged Riplet expression vectors. 24 h after transfection, cells were lysed, and WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected by western blotting with the indicated antibodies.


B ) HEK293FT were transfected with FLAG - tagged LGP2 expression vector . 24 h after transfection , immunoprecipitation was performed with anti - FLAG antibody control IgG . Immunoprecipitates were subjected to SDS - PAGE , were detected by western blotting with indicated antibodies .

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

B) HEK293FT cells were transfected with FLAG-tagged LGP2 expression vector. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody and control IgG. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies.


C ) HEK293FT were transfected with FLAG - tagged LGP2 , Riplet , HA - tagged ubiquitin expression vectors , immunoprecipitation was performed with anti - FLAG antibody .

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

C) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and HA-tagged ubiquitin expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.


D ) HEK293FT were transfected with FLAG - tagged LGP2 , Riplet , myc - tagged K63 - only - Ub expression vectors . 24 h after transfection , immunoprecipitation was performed with anti - FLAG antibody .

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

D) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and myc-tagged K63-only-Ub expression vectors. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody.


E ) WT Riplet KO HEK293 were transfected with FLAG - tagged LGP2 myc - tagged K63 - only - Ub expression vectors , immunoprecipitation was performed with anti - FLAG antibody .

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

E) WT and Riplet KO HEK293 cells were transfected with FLAG-tagged LGP2 and myc-tagged K63-only-Ub expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.


( F , G ) WT Riplet KO HEK293 were transfected with 200 ng / ml of short poly I : C for 6 h , PLA was performed with anti - LGP2 anti - K63 - Ub antibodies . number of PLA signals were counted ( t - , * p < 0 . 05 , n = 100 ) . Each dot represents number of PLA signals in each . Scale bars represent 10 μm . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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-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 O O O O O O O O O O O O O O O O O O O O O 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, G) WT and Riplet KO HEK293 cells were transfected with 200 ng/ml of short poly I:C for 6 h, and PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 100). Each dot represents the number of PLA signals in each cell. Scale bars represent 10 μm. Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( A ) Liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) of LGP2 in Riplet - expressing . LC - MS / MS data was quantified using LFQ method . chromatographic profiles for each ubiquitination site are shown in right panels . Schematic representation of ubiquitination sites of RIG - I LGP2 are shown in upper panel .

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

(A) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of LGP2 in Riplet-expressing cells. LC-MS/MS data was quantified using LFQ method. The chromatographic profiles for each ubiquitination site are shown in the right panels. Schematic representation of ubiquitination sites of RIG-I and LGP2 are shown in the upper panel.


( B ) FLAG - tagged LGP2 fragments HA - tagged Riplet expression vectors were transfected into HEK293FT . 24 h after transfection WCE were prepared . immunoprecipitation was performed with anti - FLAG antibody . Immunoprecipitates were subjected to SDS - PAGE , were detected by western blotting with indicated antibodies . data is a representative of two independent experiments .

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

(B) FLAG-tagged LGP2 fragments and HA-tagged Riplet expression vectors were transfected into HEK293FT cells. 24 h after transfection WCE were prepared. immunoprecipitation was performed with anti-FLAG antibody. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies. The data is a representative of two independent experiments.


( C ) FLAG - tagged LGP2 FLAG - tagged LGP2 - 4KR expression vectors were transfected into HEK293FT . 24 h after transfection , WCE were prepared . Immunoprecipitation was performed with anti - FLAG antibody . data is a representative of two independent experiments .

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

(C) FLAG-tagged LGP2 and FLAG-tagged LGP2-4KR expression vectors were transfected into HEK293FT cells. 24 h after transfection, WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody. The data is a representative of two independent experiments.


( A ) RIG - I , WT LGP2 , KR mutants of LGP2 were transfected into HEK293 together with p125luc reporter plasmids . Luciferase activities were determined 24 h after transfection . data represent mean ± SD ( n = 3 , t - , * p < 0 . 05 ) . WCEs were subjected to SDS - PAGE , were detected by western blotting ( lower panels ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

(A) RIG-I, WT LGP2, and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc reporter plasmids. Luciferase activities were determined 24 h after transfection. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). WCEs were subjected to SDS-PAGE, and the proteins were detected by western blotting (lower panels). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( B ) HE293 stably expressing GFP ( control ) , LGP2 , or LGP2 - 4KR were transfected with p125luc reporter plasmids then stimulated with indicated amounts of short poly I : C . WCEs were prepared 24 h after transfection stimulation , luciferase activity was determined . were subjected to SDS - PAGE detected by western blotting with indicated Abs . data represent mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

(B) HE293 cells stably expressing GFP (control), LGP2, or LGP2-4KR were transfected with p125luc reporter plasmids and then stimulated with indicated amounts of short poly I:C. WCEs were prepared 24 h after transfection and stimulation, and luciferase activity was determined. The proteins were subjected to SDS-PAGE and detected by western blotting with the indicated Abs. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 stably expressing GFP , LGP2 , or LGP2 - 4KR were transfected with 200 ng / ml of short poly I : C ( C - E ) , Total was isolated at indicated time points . expression of IFN - β , IP - 10 , Ccl5 mRNA was determined by RT - qPCR normalized to GAPDH . data represent mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

HEK293 cells stably expressing GFP, LGP2, or LGP2-4KR were transfected with 200 ng/ml of short poly I:C (C-E), Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 stably expressing LGP2 , or LGP2 - 4KR were infected with SeV at MOI = 5 ( F - H ) , Total was isolated at indicated time points . expression of IFN - β , IP - 10 , Ccl5 mRNA was determined by RT - qPCR normalized to GAPDH . data represent mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

HEK293 cells stably expressing LGP2, or LGP2-4KR were infected with SeV at MOI = 5 (F-H), Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10, and Ccl5 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


HEK293 stably expressing LGP2 , or LGP2 - 4KR were infected with influenza A ( Flu ) at MOI = 10 ( I , J ) . Total was isolated at indicated time points . expression of IFN - β , IP - 10 mRNA was determined by RT - qPCR normalized to GAPDH . data represent mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

HEK293 cells stably expressing LGP2, or LGP2-4KR were infected with influenza A virus (Flu) at MOI =10 (I, J). Total RNA was isolated at the indicated time points. The expression of IFN-β, IP-10 mRNA was determined by RT-qPCR and normalized to GAPDH. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05 Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


( K , L ) HEK293 clones stably expressing LGP2 or LGP2 - 4KR at lower were transfected with short poly I : C , expression of mRNAs were determined by RT - qPCR . data represent mean ± SD ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

(K, L) HEK293 cell clones stably expressing LGP2 or LGP2-4KR at lower levels were transfected with short poly I:C, and the expression of mRNAs were determined by RT-qPCR. The data represent the mean ± SD (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


A ) Microarray was performed as described in materials methods . A of microarray data of LGP2 LGP2 - 4KR stably expressing stimulated with short poly I : C for 12 h .

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

A) Microarray analysis was performed as described in materials and methods. A heatmap of the microarray data of LGP2 and LGP2-4KR stably expressing cells stimulated with short poly I:C for 12 h.


B , LGP2 LGP2 - 4KR stably expressing HEK293 were stimulated with 200 ng / ml of short poly I : C , expression of genes at indicated time points were determined by RT - qPCR normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

O O B-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

B, LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C ) LGP2 LGP2 - 4KR stably expressing HEK293 were stimulated with 200 ng / ml of short poly I : C , expression of genes at indicated time points were determined by RT - qPCR normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

C) LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


LGP2 LGP2 - 4KR stably expressing A549 were infected with influenza A ( Flu ) at MOI = 10 , expression of genes at indicated time points were determined by RT - qPCR normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

B-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

LGP2 and LGP2-4KR stably expressing A549 cells were infected with influenza A virus (Flu) at MOI = 10, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


H , I ) WT indicated mutant HEK293 were stimulated with 200 ng / ml of short poly I : C for 8 h . WCEs were prepared subjected to SDS - PAGE . were detected with indicated antibodies .

O O O O O O O O O O O O O O O O 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) WT and indicated mutant HEK293 cells were stimulated with 200 ng/ml of short poly I:C for 8 h. WCEs were prepared and subjected to SDS-PAGE. The proteins were detected with indicated antibodies.


J , K ) LGP2 LGP2 - 4KR expressing vectors were transected into HeLa with Riplet expressing vector . 18 hr after transfection , were fixed , then PLA signals of LGP2 endogenous MAVS were detected . Peroxisome was visualized using CellLight Peroxisome - GFP reagent . Colocalization of GFP ( peroxisome ) PLA signals ( red ) were observed by confocal microscopy . Scale bars represent 10 μm ( J ) . colocalization was normalized to of PLA signals ( n = 25 , t - , * p < 0 . 05 ) ( K ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

J, K) LGP2 and LGP2-4KR expressing vectors were transected into HeLa cells with Riplet expressing vector. 18 hr after transfection, cells were fixed, and then the PLA signals of LGP2 and endogenous MAVS were detected. Peroxisome was visualized using CellLight Peroxisome-GFP reagent. Colocalization of GFP (peroxisome) and the PLA signals (red) were observed by confocal microscopy. Scale bars represent 10 μm (J). The colocalization area was normalized to the area of the PLA signals (n = 25, t-test, *p < 0.05) (K). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


WT , Riplet KO HEK293 were stimulated with 200 ng / ml of short poly I : C . expression of SP100 , PML , ANKRD1 mRNA was determined by RT - qPCR normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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 B-MEASURED_VAR 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

WT, and Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


B ) WT , LGP2 KO , HEK293 were stimulated with 200 ng / ml of short poly I : C . expression of SP100 , PML , ANKRD1 mRNA was determined by RT - qPCR normalized to GAPDH ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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 B-MEASURED_VAR O B-MEASURED_VAR 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

B) WT, LGP2 KO, HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


C , WT Riplet KO A549 ( C ) were stimulated with 200 ng / ml of short poly I : C . expression of SP100 was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 005 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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 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

C, WT and Riplet KO A549 (C) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


D ) WT Riplet KO MEF ( D ) were stimulated with 200 ng / ml of short poly I : C . expression of SP100 was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 005 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

D) WT and Riplet KO MEF (D) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


E ) WT Riplet KO BMM were infected with influenza A ( Flu ) at MOI = 10 . PML SP100 mRNA expression was determined by RT - qPCR ( n = 3 , two - way ANOVA , * p < 0 . 05 ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

E) WT and Riplet KO BMM were infected with influenza A virus (Flu) at MOI = 10. PML and SP100 mRNA expression was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


F ) WT Riplet KO A549 were infected with influenza A infection at MOI = 10 . WCE was prepared at indicated time points subjected to SDS - PAGE . were detected by western blotting with indicated antibody .

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

F) WT and Riplet KO A549 cells were infected with influenza A virus infection at MOI = 10. WCE was prepared at the indicated time points and subjected to SDS-PAGE. Proteins were detected by western blotting with the indicated antibody.


G , LGP2 or LGP2 - 4KR stably expressing WT or Riplet KO HEK293 were infected with SeV ( G ) IFN - β mRNA at indicated time points were determined by RT - qPCR . ( n = 3 , two - way ANOVA , * p < 0 . 05 , ns : not significant ( p > 0 . 05 ) ) . Right graph in panel G is an enlargement of Y - axis of left graph ( dotted ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

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

G, LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were infected with SeV (G) IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Right graph in panel G is an enlargement of Y-axis of the left graph (dotted line). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


H ) LGP2 or LGP2 - 4KR stably expressing WT or Riplet KO HEK293 were stimulated with 200 ng / ml of short poly I : C ( H ) . IFN - β mRNA at indicated time points were determined by RT - qPCR . ( n = 3 , two - way ANOVA , * p < 0 . 05 , ns : not significant ( p > 0 . 05 ) ) . Data information : data are representative at least two independent experiments , " n " represents number of samples ( biological replicates ) . Error bars represent standard deviation from mean .

O O B-CONTROLLED_VAR O B-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 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 O

H) LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C (H). IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.


A - F ( A , B , D - F ) determined by RT - qPCR . Results are presented as mean ± SD , n = 10 . P - values are indicated on top of each plot . ( C ) Spearman correlation suggests an in vivo positive correlation between expressions of H19 Igf1r in a statistically significant manner . Spearman correlation coefficient , P - values , sample numbers are marked on top left of plot .

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

A-F (A, B, D-F) RNA levels determined by RT-qPCR. Results are presented as mean ± SD,n = 10. P-values are indicated on the top of each plot. (C) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner. Spearman correlation coefficient, P-values, and sample numbers are marked on the top left of the plot.


A - D Endometrial stromal from # 166 # 80 were each transfected with a mixture of siCon ( control siRNA ) microRNA inhibitor control ( iCon ) , siH19 iCon , or siH19 iLet7 ( let - 7 - specific inhibitor ) . were analyzed at 48 h post - transfection . Combined results from two are presented .

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

A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). RNA and proteins were analyzed at 48 h post-transfection. Combined results from the two patient cells are presented.


A - D Endometrial stromal from # 166 # 80 were each transfected with a mixture of siCon ( control siRNA ) microRNA inhibitor control ( iCon ) , siH19 iCon , or siH19 iLet7 ( let - 7 - specific inhibitor ) . Western blot gels from # 166 are shown in ( C ) . Quantitation of Western blots combining both are shown in ( D ) .

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

A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). Western blot gels from #166 cells are shown in (C). Quantitation of Western blots combining both patient cells are shown in (D).


E - H Endometrial stromal from # 98 # 212S were each transfected with empty vector or pH19 . were analyzed 48 h post - transfection . Combined results from two are presented .

O O O O O O O O O O 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 Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented.


E - H Endometrial stromal from # 98 # 212S were each transfected with empty vector or pH19 . were analyzed 48 h post - transfection . Combined results from two are presented . Western blot gels from # 98 are shown in ( G ) . Quantitation of Western blots combining both are shown in ( H ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented. Western blot gels from #98 cells are shown in (G). Quantitation of Western blots combining both patient cells are shown in (H).


A - F indicated were transfected with siCon , siH19 , empty vector , or pH19 . , viability ( as indicated by viable numbers ) , caspase activity ( as a readout for apoptosis ) were analyzed 48 h post - transfection . Combined results from two in each group are presented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented.


G E2 stimulates H19 expression in endometrial stromal . Endometrial stromal # 166 # 98 were stimulated with E2 ( + ) or vehicle ( − ) for 48 h , followed by RT - qPCR of extracted from . Results combining two are shown .

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

G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT-qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.


( B ) P2 MRI ( axial , sagittal coronal plane ) showing ventriculomegaly ( red asterisk ) , slightly thickened cortex bilateral enlarged gyri ( asterisk ) .

O O O O O 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) P2 MRI images (axial, sagittal and coronal plane) showing ventriculomegaly (red asterisk), slightly thickened cortex and bilateral enlarged gyri (white asterisk).


( D ) FIBCD1 expression in various human visceral tissues brain regions ( inset ) . Expression is plotted relative to tissue with lowest detectable expression ( trachea ; inset , choroid plexus ) . n represents technical replicates ( n = 2 ) .

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

(D) FIBCD1 expression in various human visceral tissues and brain regions (inset). Expression is plotted relative to the tissue with lowest detectable expression (trachea; inset, choroid plexus). n represents technical replicates (n = 2).


( A ) Immunofluorescent of control neuronal ( Nsyb ) CG10359 ( dFibcd1 ) RNAi - mediated knockdown D . melanogaster , 3rd instar larvae NMJ ( NMJ6 / 7 ) stained with anti - horseradish peroxidase antibodies . Empty control RNAi - mediated knockdown of CG10359 ( dFibcd1 - i ) lines # 1 , 2 3 shown . Scale bar = 20µm . Representative of 3 independent experiments . of ( A ) , control CG10359 knockdown lines NMJ neuron bouton number ( B ) n ( empty ) = 12 ; n ( # 1 ) = 20 ; n ( # 2 ) = 11 ; n ( # 3 ) = 14 . Data information : each data point represents an NMJ Data is represented as mean error bars represent . p values were calculated using 2 - way ANOVA ( panels B * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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 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 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

(A) Immunofluorescent images of control and neuronal (Nsyb) CG10359 (dFibcd1) RNAi-mediated knockdown D. melanogaster, 3rd instar larvae NMJ (NMJ6/7) stained with anti-horseradish peroxidase antibodies. Empty control and RNAi-mediated knockdown of CG10359 (dFibcd1-i) lines #1, 2 and 3 shown. Scale bar = 20µm. Representative images of 3 independent experiments. Quantification of (A), control and CG10359 knockdown lines NMJ neuron bouton number (B) n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


of NMJ neuron axon branch points ( C ) . n ( empty ) = 12 ; n ( # 1 ) = 20 ; n ( # 2 ) = 11 ; n ( # 3 ) = 14 . Data information : each data point represents an NMJ Data is represented as mean error bars represent . p values were calculated using 2 - way ANOVA ( panels C ) , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

Quantification of NMJ neuron axon branch points (C). n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels C), * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


( E ) Representative coronal section of Golgi - Cox of Fibcd1 WT KO hippocampi ( left ) , Neurolucida tracing of hippocampal CA1 pyramidal neurons ( middle ) apical dendrites with spines ( right ) . Scale bars as indicated .

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

(E) Representative coronal section images of Golgi-Cox staining of Fibcd1 WT and KO hippocampi (left), Neurolucida tracing of hippocampal CA1 pyramidal neurons (middle) and apical dendrites with spines (right). Scale bars as indicated.


( F - I ) Quantifications of ( F ) dendritic spine density , ( G ) total of apical as well as basal dendrites , ( H ) dendritic nodes in apical as well as basal dendrites ( I ) number of dendrites ( n = 5 ) . Data information : for panels F - I , each data point represents an mouse . Data is represented as mean error bars represent . p values were calculated using unpaired Student ' s t - ( panels F - I , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Quantifications of (F) dendritic spine density, (G) total length of apical as well as basal dendrites, (H) dendritic nodes in apical as well as basal dendrites and (I) number of dendrites (n = 5). Data information: for panels F-I, each data point represents an individual mouse. Data is represented as mean and error bars represent SEM. p values were calculated using unpaired Student's t-test (panels F-I, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


( B ) Immunoblot ( left ) of signal ( right ) of Fibcd1 WT ( blue ) versus Fibcd1 KO ( red ) adult hippocampi with antibodies against CS - 0S , CS - 4S , CS - 6S actin as a loading control . Each lane represents an independent ( n = 3 ) . marker sizes are indicated . Data information : For panel B , each data point represent hippocampal isolates from an mouse , Data are shown as mean values + / - . p values were calculated using paired Student ' s t - ( panels B * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis (left) and quantification of signal intensity (right) of Fibcd1 WT (blue) versus Fibcd1 KO littermates (red) adult hippocampi with antibodies against CS-0S, CS-4S, CS-6S and actin as a loading control. Each lane represents an independent animal (n = 3). Protein marker sizes are indicated. Data information: For panel B, each data point represent hippocampal protein isolates from an individual mouse, Data are shown as mean values +/- SEM. p values were calculated using paired Student's t-test (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


( F ) Confocal depicting internalisation of FITC - tagged CS - 4S by FIBCD1 - overexpressing HEK293T lines compared to untransduced unstained . Left , representative ; right , . Data is plotted as total per condition ( n = 5 ) . are further stained with CellMask Orange ( ) Hoechst ( nuclei ) . Scale bar = 50µm . Data information : for panels F , each data point represents an preparation . Data are shown as mean values + / - . p values were calculated using 1 - way ANOVA ( panels F , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

(F) Confocal images depicting internalisation of FITC-tagged CS-4S by FIBCD1-overexpressing HEK293T lines compared to untransduced cells and unstained cells. Left, representative images; right, quantification. Data is plotted as total puncta per condition (n = 5). Cells are further stained with CellMask Orange (cellular membrane) and Hoechst (nuclei). Scale bar = 50µm. Data information: for panels F, each data point represents an individual cell preparation. Data are shown as mean values +/- SEM. p values were calculated using 1-way ANOVA (panels F, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.