( G ) HAT inhibition by curcumin prevents the aberrant increase in H3K56ac and H3K27ac levels .

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(G) HAT inhibition by curcumin prevents the aberrant increase in H3K56ac and H3K27ac levels.


( H ) Violin plots depicting the distributions of the grey - scale nuclear intensity of the indicated markers .

(H) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers.


( I ) HAT inhibition prevents DHS - induced DNA damage as shown here by staining for γH2A . X ( green ) on human cardiomyocytes .

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(I) HAT inhibition prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) on human cardiomyocytes.


( J ) Quantification of γH2A . X + hCMs nuclei represented as percentage bar graph .

(J) Quantification of γH2A.X+ hCMs nuclei represented as percentage bar graph.


( K ) Curcumin - mediated HAT inhibition lead to inactivation of p21 in hCMs .

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(K) Curcumin-mediated HAT inhibition lead to inactivation of p21 in hCMs.


( L ) Violin plots depicting the distributions of the grey - scale nuclear intensity of p21 stained nuclei . ( When not specified , the experiments were conducted in at least 3 biological replicates ) .

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(L) Violin plots depicting the distributions of the grey-scale nuclear intensity of p21 stained nuclei. (When not specified, the experiments were conducted in at least 3 biological replicates).


( D ) Heatmap depicting the expression profile of the markers of DNA damage response in the indicated conditions .

(D) Heatmap depicting the expression profile of the markers of DNA damage response in the indicated conditions.


( E ) Co - treatment of curcumin activates DNA repair pathways as depicted here by heatmaps .

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(E) Co-treatment of curcumin activates DNA repair pathways as depicted here by heatmaps.


Representative micrographs depicting ( B ) γH2A . X staining on the ventricle sections for the indicated cohorts .

Representative micrographs depicting (B) γH2A.X staining on the ventricle sections for the indicated cohorts.


Representative micrographs depicting ( C ) H3K27ac staining on the ventricle sections for the indicated cohorts .

Representative micrographs depicting (C) H3K27ac staining on the ventricle sections for the indicated cohorts.


( D ) Quantification of γH2A . X + CMs nuclei represented as percentage bar graph .

(D) Quantification of γH2A.X+ CMs nuclei represented as percentage bar graph.


( E ) Box - plot depicting the distributions of the corrected total nuclear fluorescence of the indicated cohorts .

(E) Box-plot depicting the distributions of the corrected total nuclear fluorescence of the indicated cohorts.


( F ) Representative micrographs depicting ventricular view from the echocardiography of the indicated cohorts .

(F) Representative micrographs depicting ventricular view from the echocardiography of the indicated cohorts.


( G - H ) Quantification of fractional shortening and ejection fraction of the indicated groups represented as bean plot and percentage bar graph respectively ( n = 6 animals per condition ) . Scale bars for B , C = 50 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

(G-H) Quantification of fractional shortening and ejection fraction of the indicated groups represented as bean plot and percentage bar graph respectively (n=6 animals per condition). Scale bars for B,C = 50 µm.


The percentage of dead cells was determined at the indicated timepoints by trypan blue exclusion assay ( mean ± s . d . ; n = 3 ) . ( a ) Bif - 1 + / + ( wild type ; WT ) and Bif - 1 − / − ( knockout ; KO ) MEFs were cultured in EBSS for 0 , 3 , 6 or 12 h . ( b ) Bif - 1 − / − MEFs stably transfected with a Bif - 1 expression plasmid or control empty vector were incubated in EBSS for 0 or 12 h . Recovery of Bif - 1 expression in Bif - 1 − / − cells was determined by immunoblot analysis ( inset ) .

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The percentage of dead cells was determined at the indicated timepoints by trypan blue exclusion assay (mean ± s.d.; n = 3). (a) Bif-1+/+ (wild type; WT) and Bif-1−/− (knockout; KO) MEFs were cultured in EBSS for 0, 3, 6 or 12 h. (b) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were incubated in EBSS for 0 or 12 h. Recovery of Bif-1 expression in Bif-1−/− cells was determined by immunoblot analysis (inset).


( c ) Bif - 1 + / + and Bif - 1 − / − MEFs were cultured in EBSS in the presence of 50 μM z - VAD - fmk or control DMSO for 0 , 3 , 6 or 12 h .

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(c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h.


( d ) Bax + / + Bak + / + ( WT ) and Bax − / − Bak − / − ( DKO ) MEFs were cultured in EBSS in the presence of 50 μM z - VAD - fmk or control DMSO for 0 , 6 or 12 h .

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(d) Bax+/+Bak+/+ (WT) and Bax−/−Bak−/− (DKO) MEFs were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 6 or 12 h.


( a , b ) Bif - 1 + / + and Bif - 1 − / − MEFs or Bax + / + Bak + / + and Bax − / − Bak − / − MEFs were infected with Beclin 1 shRNA or control shRNA ( SCR shRNA ) lentiviruses . Twenty - four hours after infection , the medium was replaced with fresh culture medium and the cells were allowed to recover for an additional 48 h . The cells were then cultured in EBSS for 0 , 6 or 12 h , and the percentage of dead cells was determined by trypan blue exclusion assay ( mean ± s . d . ; n = 3 ) . Knockdown of Beclin 1 expression was confirmed by immunoblot analysis ( insets ) .

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(a, b) Bif-1+/+ and Bif-1−/− MEFs or Bax+/+Bak+/+ and Bax−/−Bak−/− MEFs were infected with Beclin 1 shRNA or control shRNA (SCR shRNA) lentiviruses. Twenty-four hours after infection, the medium was replaced with fresh culture medium and the cells were allowed to recover for an additional 48 h. The cells were then cultured in EBSS for 0, 6 or 12 h, and the percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Knockdown of Beclin 1 expression was confirmed by immunoblot analysis (insets).


( c , d ) Bif - 1 + / + and Bif - 1 − / − MEFs were pre - treated with 10 mM 3 - methyladenine ( 3 - MA ) , 0 . 2 μM wortmannin ( WM ) or control DMSO for 30 min followed by culture in EBSS for the indicated times . The percentage of dead cells was determined by trypan blue exclusion assay ( mean ± s . d . ; n = 3 ) .

O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).


( c , d ) Bif - 1 + / + and Bif - 1 − / − MEFs were pre - treated with 10 mM 3 - methyladenine ( 3 - MA ) , 0 . 2 μM wortmannin ( WM ) or control DMSO for 30 min followed by culture in EBSS for the indicated times . The percentage of dead cells was determined by trypan blue exclusion assay ( mean ± s . d . ; n = 3 ) . Immunoblot analyses were performed to detect LC3 modification and caspase - 3 activation .

O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bif-1+/+ and Bif-1−/− MEFs were pre-treated with 10 mM 3-methyladenine (3-MA), 0.2 μM wortmannin (WM) or control DMSO for 30 min followed by culture in EBSS for the indicated times. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3). Immunoblot analyses were performed to detect LC3 modification and caspase-3 activation.


( e ) Atg5 + / + and Atg5 − / − MEFs were cultured in EBSS in the presence of 50 μM of z - VAD - fmk or control DMSO for 0 , 6 or 12 h . The percentage of dead cells was determined by trypan blue exclusion assay ( mean ± s . d . ; n = 3 ) .

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(e) Atg5+/+ and Atg5−/− MEFs were cultured in EBSS in the presence of 50 μM of z-VAD-fmk or control DMSO for 0, 6 or 12 h. The percentage of dead cells was determined by trypan blue exclusion assay (mean ± s.d.; n = 3).


( f ) Total cell lysates ( TCL ) were prepared from Atg5 + / + and Atg5 − / − MEFs cultured in EBSS for the indicated times and subjected to immunoblot analyses . Uncropped images of the immunoblots in a , b , d and f are shown in the Supplementary Information , Fig . S5 .

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(f) Total cell lysates (TCL) were prepared from Atg5+/+ and Atg5−/− MEFs cultured in EBSS for the indicated times and subjected to immunoblot analyses. Uncropped images of the immunoblots in a, b, d and f are shown in the Supplementary Information, Fig. S5.


( a ) Bif - 1 + / + and Bif - 1 − / − MEFs were cultured in complete medium ( CM ) or EBSS for 3 h and analysed by transmission electron microscopy . Arrows indicate autophagosomes . The number of autophagosomes per cross - sectioned cell was counted ( mean ± s . d . ; n = 42 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bif-1+/+ and Bif-1−/− MEFs were cultured in complete medium (CM) or EBSS for 3 h and analysed by transmission electron microscopy. Arrows indicate autophagosomes. The number of autophagosomes per cross-sectioned cell was counted (mean ± s.d.; n = 42).


( b ) Bif - 1 + / + and Bif - 1 − / − MEFs were transfected with GFP - LC3 , cultured in LC3 or complete medium for 3 h , then analysed by fluorescent microscopy . The number of GFP - LC3 dots per GFP - positive cell was counted ( mean ± s . d . ; n = 35 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bif-1+/+ and Bif-1−/− MEFs were transfected with GFP-LC3, cultured in LC3 or complete medium for 3 h, then analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 35).


( c ) Bif - 1 + / + and Bif - 1 − / − MEFs were cultured in EBSS for the indicated times and subjected to immunoblot analysis with anti - LC3 , anti - α - tubulin and anti - β - actin antibodies .

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(c) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for the indicated times and subjected to immunoblot analysis with anti-LC3, anti-α-tubulin and anti-β-actin antibodies.


( d ) Bif - 1 − / − MEFs stably transfected with a Bif - 1 expression plasmid or control empty vector were cultured in EBSS for 0 , 3 or 6 h and subjected to immunoblot analysis . The immunoblotting results of LC3 in c and d were quantified as the percentage of LC3 - II out of total LC3 ( LC3 - I + LC3 - II ) and are represented in bar graphs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bif-1−/− MEFs stably transfected with a Bif-1 expression plasmid or control empty vector were cultured in EBSS for 0, 3 or 6 h and subjected to immunoblot analysis. The immunoblotting results of LC3 in c and d were quantified as the percentage of LC3-II out of total LC3 (LC3-I + LC3-II) and are represented in bar graphs.


( e ) Stable Bif - 1 shRNA - expressing clones or control HeLa cells transfected with GFP - LC3 were cultured in complete medium or EBSS for 2 h and analysed by fluorescent microscopy . The number of GFP - LC3 dots per GFP - positive cell was quantified ( mean ± s . d . ; n = 66 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stable Bif-1 shRNA-expressing clones or control HeLa cells transfected with GFP-LC3 were cultured in complete medium or EBSS for 2 h and analysed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 66).


( f ) Bif - 1 knockdown HeLa cells were transfected with GFP - LC3 in combination with Bif - 1 shRNA - resistant Bif - 1 - HcRed , Bif - 1ΔBAR - HcRed or empty HcRed vector for 20 h . The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP - LC3 dots per HcRed - positive cell was determined using a fluorescence microscope ( mean ± s . d . ; n = 35 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bif-1 knockdown HeLa cells were transfected with GFP-LC3 in combination with Bif-1 shRNA-resistant Bif-1-HcRed, Bif-1ΔBAR-HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 35).


( a ) COS7 cells were transfected with Bif - 1 - HcRed together with GFP - Atg5 or GFP - LC3 . Twenty hours after transfection , the cells were cultured in EBSS ( middle and bottom panels ) or complete medium ( top panel ) for 2 h and subjected to confocal microscopy analysis . Magnified images are shown as insets . Representative Bif - 1 - Atg5 or Bif - 1 - LC - 3 double - positive foci are indicated by arrows .

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(a) COS7 cells were transfected with Bif-1-HcRed together with GFP-Atg5 or GFP-LC3. Twenty hours after transfection, the cells were cultured in EBSS (middle and bottom panels) or complete medium (top panel) for 2 h and subjected to confocal microscopy analysis. Magnified images are shown as insets. Representative Bif-1-Atg5 or Bif-1-LC-3 double-positive foci are indicated by arrows.


( b ) COS7 cells transfected with Bif - 1 - GFP were cultured in EBSS for 2 h and the localization of Bif - 1 - GFP was examined by immunogold electron microscopy using anti - GFP antibodies . Arrows indicate representative gold particles that are detected on autophagosomal membranes . The scale bars represent 10 μm in the main images and 1 μm in the insets in a , and 1 μm in b .

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(b) COS7 cells transfected with Bif-1-GFP were cultured in EBSS for 2 h and the localization of Bif-1-GFP was examined by immunogold electron microscopy using anti-GFP antibodies. Arrows indicate representative gold particles that are detected on autophagosomal membranes. The scale bars represent 10 μm in the main images and 1 μm in the insets in a, and 1 μm in b.


( a ) Bif - 1 + / + and Bif - 1 − / − MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti - Bif - 1 monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with the indicated antibodies .

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(a) Bif-1+/+ and Bif-1−/− MEFs were cultured in EBSS for 0 or 6 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with the indicated antibodies.


( b ) 293T cells were cotransfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti - Flag monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Myc or anti - Flag polyclonal 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 O O O O O O

(b) 293T cells were cotransfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies.


( c ) 293T cells were cotransfected with the indicated plasmids for 22 h , starved in EBSS for 2 h and subjected to immunoprecipitation with anti - Flag monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Myc or anti - Flag polyclonal antibodies .

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(c) 293T cells were cotransfected with the indicated plasmids for 22 h, starved in EBSS for 2 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc or anti-Flag polyclonal antibodies.


( d ) 293T cells were transfected with GFP siRNA or UVRAG siRNA for 24 h , starved in 2 h for 2 h and subjected to immunoprecipitation with anti - Bif - 1 monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Bif - 1 and Beclin 1 polyclonal antibodies . The reduction of UVRAG protein expression by UVRAG siRNA was confirmed by immunoprecipitation and immunoblot analysis with anti - UVRAG polyclonal antibody . Uncropped images of the immunoblots are shown in the Supplementary Information , Fig . S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were transfected with GFP siRNA or UVRAG siRNA for 24 h, starved in 2 h for 2 h and subjected to immunoprecipitation with anti-Bif-1 monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Bif-1 and Beclin 1 polyclonal antibodies. The reduction of UVRAG protein expression by UVRAG siRNA was confirmed by immunoprecipitation and immunoblot analysis with anti-UVRAG polyclonal antibody. Uncropped images of the immunoblots are shown in the Supplementary Information, Fig. S5.


( a ) HeLa cells were transfected with 0 . 2 μg GFP - LC3 and 0 , 0 . 2 or 0 . 6 μg of Bif - 1SH3 - Myc expression plasmids for 24 h . The total amount of plasmid DNA was adjusted at 0 . 8 μg per transfection with the pcDNA3 vector . The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP - LC3 dots per GFP - positive cell was determined using a fluorescence microscope ( mean ± s . d . ; n = 40 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with 0.2 μg GFP-LC3 and 0, 0.2 or 0.6 μg of Bif-1SH3-Myc expression plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg per transfection with the pcDNA3 vector. The cells were then cultured in EBSS or complete medium for 3 h and the number of GFP-LC3 dots per GFP-positive cell was determined using a fluorescence microscope (mean ± s.d.; n = 40).


( b ) HeLa cells were transfected with GFP - LC3 in combination with either Bif - 1SH3 - HcRed , Endophilin A1SH3 - HcRed or empty HcRed vector for 20 h . The cells were then cultured in 2 h or complete medium for 2 h and the number of GFP - LC3 dots per HcRed - positive cell was counted using a fluorescence microscope ( mean ± s . d . ; n = 46 ) . Statistical significance in a and b was determined by Student ' s t - test and the asterisks indicate P 0 . 0001 .

(b) HeLa cells were transfected with GFP-LC3 in combination with either Bif-1SH3-HcRed, Endophilin A1SH3-HcRed or empty HcRed vector for 20 h. The cells were then cultured in 2 h or complete medium for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was counted using a fluorescence microscope (mean ± s.d.; n = 46). Statistical significance in a and b was determined by Student's t-test and the asterisks indicate P 0.0001.


( c ) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti - Flag monoclonal antibodies . The resulting immune complexes and TCL were analysed by immunoblotting with anti - Myc and anti - Flag polyclonal antibodies . Uncropped images of the immunoblots in c are shown in the Supplementary Information , Fig . S5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibodies. The resulting immune complexes and TCL were analysed by immunoblotting with anti-Myc and anti-Flag polyclonal antibodies. Uncropped images of the immunoblots in c are shown in the Supplementary Information, Fig. S5.


( a ) Bif - 1 + / + and Bif - 1 − / − MEFs were transfected with Flag - PI ( 3 ) KC3 , then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI ( 3 ) KC3 kinase assay . The expression of Flag - PI ( 3 ) KC3 was confirmed by immunoblot analysis ( left panel ) . A representative autoradiograph is shown ( middle ) . The results were quantified by phosphoimaging with a Typhoon scanner and shown as fold activation ( right ) . The data are presented as means ± s . d . of five independent experiments .

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(a) Bif-1+/+ and Bif-1−/− MEFs were transfected with Flag-PI(3)KC3, then incubated in complete medium or EBSS for 2 h and subjected to an in vitro PI(3)KC3 kinase assay. The expression of Flag-PI(3)KC3 was confirmed by immunoblot analysis (left panel). A representative autoradiograph is shown (middle). The results were quantified by phosphoimaging with a Typhoon scanner and shown as fold activation (right). The data are presented as means ± s.d. of five independent experiments.


( b ) Bif - 1 + / + and Bif - 1 − / − MEFs were transfected with p40 ( phox ) PX - EGFP , cultured in complete medium or EBSS for 2 h and analysed using a fluorescence microscope . The number of p40 ( phox ) PX - EGFP - positive vesicles per cell was counted ( mean ± s . d . ; n = 35 ) . Statistical significance in a and b was determined by Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bif-1+/+ and Bif-1−/− MEFs were transfected with p40(phox) PX-EGFP, cultured in complete medium or EBSS for 2 h and analysed using a fluorescence microscope. The number of p40(phox) PX-EGFP-positive vesicles per cell was counted (mean ± s.d.; n = 35). Statistical significance in a and b was determined by Student's t-test.


( c ) Stable Bif - 1 shRNA clones or control HeLa cells were transfected with Flag - PI ( 3 ) KC3 alone or in combination with Flag - tagged Beclin 1 and UVRAG for 48 h and subjected to an in vitro PI ( 3 ) KC3 lipid kinase assay . The results were quantified and are shown as relative activities ( mean ± s . d . ; n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stable Bif-1 shRNA clones or control HeLa cells were transfected with Flag-PI(3)KC3 alone or in combination with Flag-tagged Beclin 1 and UVRAG for 48 h and subjected to an in vitro PI(3)KC3 lipid kinase assay. The results were quantified and are shown as relative activities (mean ± s.d.; n = 3).


( d ) 293T cells were cotransfected with Flag - PI ( 3 ) KC3 and either wild - type Bif - 1 , Bif - 1ΔSH3 or control empty vector for 24 h and subjected to an in vitro PI ( 3 ) KC3 kinase assay ( mean ± s . d . ; n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were cotransfected with Flag-PI(3)KC3 and either wild-type Bif-1, Bif-1ΔSH3 or control empty vector for 24 h and subjected to an in vitro PI(3)KC3 kinase assay (mean ± s.d.; n = 3).


( a ) Bif - 1 − / − mice exhibit moderate splenomegaly . The weight of spleens extirpated from Bif - 1 + / + ( n = 27 ) and Bif - 1 − / − ( n = 34 ) mice ( 6 - 18 - month old ) was measured . Statistical significance was determined by Wilcoxon rank test .

(a) Bif-1−/− mice exhibit moderate splenomegaly. The weight of spleens extirpated from Bif-1+/+ (n = 27) and Bif-1−/− (n = 34) mice (6-18-month old) was measured. Statistical significance was determined by Wilcoxon rank test.


( b ) Hematoxylin and eosin staining of tumour tissues . Tumours were observed in 3 of 21 ( 14 . 3 % ) and 26 of 29 ( 89 . 7 % ) autopsied Bif - 1 + / + and Bif - 1 − / − mice , respectively . The scale bars represent 50 μm .

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(b) Hematoxylin and eosin staining of tumour tissues. Tumours were observed in 3 of 21 (14.3%) and 26 of 29 (89.7%) autopsied Bif-1+/+ and Bif-1−/− mice, respectively. The scale bars represent 50 μm.


( B ) Confocal images of HeLa cells transfected with EGFP - ORP5 or EGFP - ORP8 showing variable expression levels . Arrows indicate ORP5 and ORP8 localization to cortical ER ( cER ) . Scale bar , 10 μm .

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(B) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 showing variable expression levels. Arrows indicate ORP5 and ORP8 localization to cortical ER (cER). Scale bar, 10 μm.


( C - D ) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP - ORP5 or EGFP - ORP8 and immunogold stained with anti - GFP ( 15 nm gold ) to detect ORP5 or ORP8 and anti - PDI ( 10 nm gold ) to label the ER lumen . ORP5 and ORP8 localize at ER - PM ( arrows ) and ER - mitochondria contact sites ( arrowheads ) . m , mitochondria ; cER , cortical ER . Scale bar 200nm . ( E - F ) Quantification of the IEM labelling for EGFP - ORP5 , EGFP - ORP8 ( E ) and GFP - Sec61β ( F ) in transfected HeLa cells . Results are presented as the percentage of the total number of gold particles ( 800 per condition , n = 35 - 45 cells ) for ORP5 , ORP8 and Sec61β in the indicated compartments ( non - cER , non - cortical ER ; ER - PM , ER - PM contact sites ; ER - mito , ER - mitochondria contact sites . % gold particles ± SEM . Data are the mean of three independent replicates . * P < 0 , 001 compared to non - cER and ER - mito [ EGFP - ORP5 ] and to ER - PM and ER - mito [ EGFP - ORP8 and GFP - Sec61β ] , # P < 0 , 001 compared to ER - PM and ER - mito [ GFP - Sec61β ] ) .

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(C-D) Electron microscopy of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 and immunogold stained with anti-GFP (15 nm gold) to detect ORP5 or ORP8 and anti-PDI (10 nm gold) to label the ER lumen. ORP5 and ORP8 localize at ER-PM (arrows) and ER-mitochondria contact sites (arrowheads). m, mitochondria; cER, cortical ER. Scale bar 200nm. (E-F) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 (E) and GFP-Sec61β (F) in transfected HeLa cells. Results are presented as the percentage of the total number of gold particles (800 per condition, n=35-45 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (non-cER, non-cortical ER; ER-PM, ER-PM contact sites; ER-mito, ER-mitochondria contact sites. % gold particles ±SEM. Data are the mean of three independent replicates. *P<0,001 compared to non-cER and ER-mito [EGFP-ORP5] and to ER-PM and ER-mito [EGFP-ORP8 and GFP-Sec61β], #P<0,001 compared to ER-PM and ER-mito [GFP-Sec61β]).


( G ) Representative electron microscopy of mitochondria and ( H ) quantification of the percentage of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa cells expressing EGFP - ORP5 , EGFP - ORP8 together with HRP - KDEL , to visualize the ER . Control ( Ctrl ) consisted of HeLa cells overexpressing only HRP - KDEL .

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(G) Representative electron microscopy of mitochondria and (H) quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells expressing EGFP-ORP5, EGFP-ORP8 together with HRP-KDEL, to visualize the ER. Control (Ctrl) consisted of HeLa cells overexpressing only HRP-KDEL.


( I ) Crude mitochondria , mitochondria and MAM fractions were purified from HeLa cells and equal amount of protein ( 25g ) from each fraction were loaded on a 4 - 20 % gradient SDS - PAGE gel and immunoblotted using anti - ORP5 , anti - ORP8 and anti - IP3R - 3 ( MAM protein ) , anti - PDI ( ER protein ) and anti - cytochrome c ( mitochondrial protein ) . TC , Total cell ; MAM , mitochondria - associated ER membrane .

(I) Crude mitochondria, mitochondria and MAM fractions were purified from HeLa cells and equal amount of protein (25g) from each fraction were loaded on a 4-20% gradient SDS-PAGE gel and immunoblotted using anti-ORP5, anti-ORP8 and anti-IP3R-3 (MAM protein), anti-PDI (ER protein) and anti- cytochrome c (mitochondrial protein). TC,Total cell; MAM, mitochondria-associated ER membrane .


( A ) Confocal images of HeLa cells transfected with EGFP - ORP5 , EGFP - ORP8 , or GFP - Sec61β together with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP8, or GFP-Sec61β together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( B ) Electron microscopy of ultrathin cryosections of HeLa cells co - transfected with EGFP - ORP5 and PTPIP51 - HA and immunogold stained with anti - GFP ( 15 nm gold ) and anti - HA ( 10 nm gold ) showing EGFP - ORP5 localization at ER - mitochondria contacts ( arrows ) . Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria . Scale bar 200 nm . ( C ) Quantification of the IEM labelling for EGFP - ORP5 , EGFP - ORP8 and GFP - Sec61β in HeLa cells co - transfected with PTPIP51 - HA . Results are presented as the percentage of the total number of gold particles ( 800 per condition , n = 30 - 35 cells ) for ORP5 , ORP8 and Sec61β in the indicated compartments ( ER , reticular ER ; ER - mito MCS , ER - mitochondria membrane contact sites ; ER - PM MCS , ER - PM membrane contact sites ) . % gold particles ± SEM . Data are representative of three independent replicates . * * P < 0 , 001 .

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(B) Electron microscopy of ultrathin cryosections of HeLa cells co-transfected with EGFP-ORP5 and PTPIP51-HA and immunogold stained with anti-GFP (15 nm gold) and anti-HA (10 nm gold) showing EGFP-ORP5 localization at ER-mitochondria contacts (arrows). Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria. Scale bar 200 nm. (C) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 and GFP-Sec61β in HeLa cells co-transfected with PTPIP51-HA. Results are presented as the percentage of the total number of gold particles (800 per condition, n=30-35 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (ER, reticular ER; ER-mito MCS, ER-mitochondria membrane contact sites; ER-PM MCS, ER-PM membrane contact sites). % gold particles ±SEM. Data are representative of three independent replicates. **P<0,001.


( D ) Confocal images of HeLa cells transfected with EGFP - ORP5 or EGFP - ORP8 together with PTPIP51 - HA and RFP - Sec61β and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

(D) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 together with PTPIP51-HA and RFP-Sec61β and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( E ) Representative EM images of ER - mitochondria contacts in the indicated overexpression conditions . ( F ) Quantification of the percentage of ER - mitochondria contact sites ( ER - mito contact ) per mitochondria of HeLa cells transfected with HRP - KDEL ( Ctrl ) or co - transfected with PTPIP51 - HA and EGFP or PTPIP51 - HA and EGFP - ORP5 or PTPIP51 - HA and EGFP - ORP8 ( n = 30 cells and 1082 - 1223 mitochondria ) . % ER - mitochondria contact sites ± SEM . Data are representative of three independent replicates . * * P < 0 , 001 , * P < 0 , 05 .

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(E) Representative EM images of ER-mitochondria contacts in the indicated overexpression conditions. (F) Quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells transfected with HRP-KDEL (Ctrl) or co-transfected with PTPIP51-HA and EGFP or PTPIP51-HA and EGFP-ORP5 or PTPIP51-HA and EGFP-ORP8 (n=30 cells and 1082-1223 mitochondria). % ER-mitochondria contact sites ±SEM. Data are representative of three independent replicates. **P<0,001, *P<0,05.


( G ) EGFP , EGFP - ORP5 or EGFP - ORP8 were immuno - precipitated from lysates of HeLa cells co - expressing PTPIP51 - HA and treated with control ( siCtrl ) or VAPA and VAPB ( SiVAPA / B ) siRNAs and analyzed by western blot using antibodies against GFP ( ORPs ) , HA ( PTPIP51 ) , VAPA , VAPB and tubulin .

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(G) EGFP, EGFP-ORP5 or EGFP-ORP8 were immuno-precipitated from lysates of HeLa cells co-expressing PTPIP51-HA and treated with control (siCtrl) or VAPA and VAPB (SiVAPA/B) siRNAs and analyzed by western blot using antibodies against GFP (ORPs), HA (PTPIP51), VAPA, VAPB and tubulin.


( A ) Confocal images of HeLa cells transfected with EGFP - ORP5 , EGFP - ORP5PH or EGFP - ORP5ORD and immunostained using anti - TOM20 antibody to visualize mitochondria . Scale bar , 10 μm .

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(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP5PH or EGFP-ORP5ORD and immunostained using anti-TOM20 antibody to visualize mitochondria. Scale bar, 10 μm.


( B ) Confocal images of HeLa cells transfected with the EGFP - tagged ORD domain of ORP5 ( EGFP - ORD ORP5 ) and immunostained using anti - TOM20 antibody ( upper panels ) or co - transfected with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(B) Confocal images of HeLa cells transfected with the EGFP-tagged ORD domain of ORP5 (EGFP-ORD ORP5) and immunostained using anti-TOM20 antibody (upper panels) or co-transfected with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( C - D ) Confocal images of HeLa cells transfected with EGFP - ORP5PH or EGFP - ORP5ORD ( C ) together with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

(C-D) Confocal images of HeLa cells transfected with EGFP-ORP5PH or EGFP-ORP5ORD (C) together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( C - D ) Confocal images of HeLa cells transfected with EGFP - ORP5 , EGFP - ORP5L389D or EGFP - ORP8L425D ( D ) together with PTPIP51 - HA and immunostained using HA antibody to detect PTPIP51 . Scale bar , 10 μm .

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(C-D) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP5L389D or EGFP-ORP8L425D (D) together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm.


( E ) EGFP - ORP5 , EGFP - ORP8 , EGFP - ORP5L389D , EGFP - ORP8L425D , GFP - Sec61β or EGFP alone were immunoprecipitated from lysates of HeLa cells co - expressing PTPIP51 - HA and analyzed by western blot with antibodies against GFP and HA .

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(E) EGFP-ORP5, EGFP-ORP8, EGFP-ORP5L389D, EGFP-ORP8L425D, GFP-Sec61β or EGFP alone were immunoprecipitated from lysates of HeLa cells co-expressing PTPIP51-HA and analyzed by western blot with antibodies against GFP and HA.


( A ) ORP5 , ORP8 and actin levels of HRP - KDEL - expressing HeLa cells treated with Ctrl siRNAs ( siCtrl ) or siRNAs against ORP5 ( siORP5 ) or ORP8 ( siORP8 ) .

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(A) ORP5, ORP8 and actin levels of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs (siCtrl) or siRNAs against ORP5 (siORP5) or ORP8 (siORP8).


( B ) Electron microscopy of HRP - KDEL - expressing HeLa cells treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8 . Red arrows indicate ER - PM contact sites . Scale bar , 2 μm . Insets show representative mitochondria and red arrows in the insets indicate ER - mitochondria contacts . Inset scale bar , 500nm .

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(B) Electron microscopy of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8. Red arrows indicate ER-PM contact sites. Scale bar, 2 μm. Insets show representative mitochondria and red arrows in the insets indicate ER-mitochondria contacts. Inset scale bar, 500nm.


( C ) Representative EM micrographs showing the morphology of mitochondria in siCtrl , siORP5 and siORP8 - treated cells . Scale bar , 1 μm . ( D ) Quantifications of the number of mitochondria with altered morphology in the indicated siRNA conditions . % altered mitochondria ± SEM , n = 25 cells and 632 - 710 mitochondria . * P < 0 , 001 compared to siCtrl .

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(C) Representative EM micrographs showing the morphology of mitochondria in siCtrl, siORP5 and siORP8-treated cells. Scale bar, 1 μm. (D) Quantifications of the number of mitochondria with altered morphology in the indicated siRNA conditions. % altered mitochondria ±SEM, n=25 cells and 632-710 mitochondria. *P<0,001 compared to siCtrl.


( E ) Mitochondrial oxygen consumption rate ( OCR ) measured in Ctrl and ORP5 siRNA transfected cells . OCR trace was obtained by sequential measurement of basal OCR ( OCRBAS ) , OCR after the addition of oligomycin ( OM ) and OCR after addition of antimycin A ( AA ) . Note the reduced basal OCR ( OCRBAS ) compared to ctrl siRNA cells . Error bars denote ± SEM . Data are the mean of 4 independent repeats ( n = 4 ) , * P < 0 , 05 .

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(E) Mitochondrial oxygen consumption rate (OCR) measured in Ctrl and ORP5 siRNA transfected cells. OCR trace was obtained by sequential measurement of basal OCR (OCRBAS), OCR after the addition of oligomycin (OM) and OCR after addition of antimycin A (AA). Note the reduced basal OCR (OCRBAS) compared to ctrl siRNA cells. Error bars denote ±SEM. Data are the mean of 4 independent repeats (n=4), *P<0,05.


( A ) expression of FUS does not alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in transfected NSC34 cells . Immunoblots of NSC34 cells transfected with EGFP as a control ( CTRL ) , or wild - type or mutant EGFP - FUS . Transfected cells were purified via EGFP using a cell sorter and the samples probed on immunoblots as indicated . On the FUS immunoblot , samples were probed with FUS antibody to show endogenous and transfected proteins ; tubulin is shown as a loading control .

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(A) expression of FUS does not alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in transfected NSC34 cells. Immunoblots of NSC34 cells transfected with EGFP as a control (CTRL), or wild-type or mutant EGFP-FUS. Transfected cells were purified via EGFP using a cell sorter and the samples probed on immunoblots as indicated. On the FUS immunoblot, samples were probed with FUS antibody to show endogenous and transfected proteins; tubulin is shown as a loading control.


( B ) representative electron microscopy of ER - mitochondria associations in NSC34 cells transfected with control EGFP vector ( CTRL ) , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K as indicated ; arrowheads with loops show regions of association . Scale bar = 200 nm . Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples . Data were analysed by one - way analysis of variance ( ANOVA ) followed by Tukey ' s multiple comparison test . N = 27 - 30 cells and 247 - 424 mitochondria , error bars are s . e . m . ; * * * p < 0 . 001 .

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(B) representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with control EGFP vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K as indicated; arrowheads with loops show regions of association. Scale bar=200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. N=27-30 cells and 247-424 mitochondria, error bars are s.e.m.; ***p<0.001.


( C and D ) siRNA loss of FUS does not affect ER - mitochondria associations or alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in NSC34 cells . ( C ) immunoblots of cells either mock transfected or treated with control ( CTRL ) or FUS siRNAs ; GAPDH is shown as a loading control .

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(C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (C) immunoblots of cells either mock transfected or treated with control (CTRL) or FUS siRNAs; GAPDH is shown as a loading control.


( C and D ) siRNA loss of FUS does not affect ER - mitochondria associations or alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in NSC34 cells . ( D ) representative electron microscopy of ER - mitochondria associations in control ( CTRL ) and FUS siRNA treated cells . Arrowheads with loops show regions of association . Scale bar = 200 nm . Data analysed by unpaired t - test . N = 27 - 28 cells and 193 - 202 mitochondria .

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(C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (D) representative electron microscopy of ER-mitochondria associations in control (CTRL) and FUS siRNA treated cells. Arrowheads with loops show regions of association. Scale bar=200 nm. Data analysed by unpaired t-test. N=27-28 cells and 193-202 mitochondria.


( A ) FU - induced reductions in ER - mitochondria associations can be detected using SIM . NSC34 cells were transfected with either EGFP control vector , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K and immunostained for TOM20 and PDI to label mitochondria ( Mito ) and ER respectively ; FUS were detected via their EGFP tags . Merge ( ZOOM ) show zoomed images of boxed regions and co - localisation shows co - localised pixels . Scale bar = 2 m . Bar chart shows ER - mitochondria co - localisation ( Manders coefficient ) normalized to control in the different samples . Data were analysed by one - way ANOVA with Tukey ' s post - hoc test . 10 - 14 cells were analysed per condition from 3 independent experiments ; error bars are s . e . m . , * * p < 0 . 01 and * * * p < 0 . 001 .

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(A) FU-induced reductions in ER-mitochondria associations can be detected using SIM. NSC34 cells were transfected with either EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and immunostained for TOM20 and PDI to label mitochondria (Mito) and ER respectively; FUS were detected via their EGFP tags. Merge (ZOOM) show zoomed images of boxed regions and co-localisation shows co-localised pixels. Scale bar=2 m. Bar chart shows ER-mitochondria co-localisation (Manders coefficient) normalized to control in the different samples. Data were analysed by one-way ANOVA with Tukey's post-hoc test. 10-14 cells were analysed per condition from 3 independent experiments; error bars are s.e.m., **p<0.01 and ***p<0.001.


( B ) ER - mitochondria associations and the VAPB - PTPIP51 interaction are disrupted by wild - type and ALS / FTD mutant FUS . NSC34 cells were transfected with EGFP control vector ( CTRL ) , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K and proximity ligation assays performed using VAPB and PTPIP51 antibodies . FUS were detected via their EGFP tags . Scale bar = 10 m . Bar chart shows relative number of proximity ligation assay signals / cell . Data were analysed by one - way ANOVA and Tukey ' s post - hoc test ; n = 47 - 53 cells from 5 experiments . Error bars are s . e . m . ; * * * p < 0 . 001 .

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(B) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted by wild-type and ALS/FTD mutant FUS. NSC34 cells were transfected with EGFP control vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and proximity ligation assays performed using VAPB and PTPIP51 antibodies. FUS were detected via their EGFP tags. Scale bar=10 m. Bar chart shows relative number of proximity ligation assay signals/cell. Data were analysed by one-way ANOVA and Tukey's post-hoc test; n=47-53 cells from 5 experiments. Error bars are s.e.m.; ***p<0.001.


( C ) overexpression of FUS reduces the binding of VAPB to PTPIP51 in transfected cells . Cells were transfected as indicated with either control empty vector ( CTRL ) , HA - PTPIP51 + CTRL , or HA - PTPIP51 + either EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K . PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting . Both inputs and immunoprecipitations ( IP ) are shown and no immunoprecipitating VAPB signals were obtained in the absence of HA - PTPIP51 . Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots . VAPB signals were normalized to immunoprecipitated PTPIP51 - HA signals .

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(C) overexpression of FUS reduces the binding of VAPB to PTPIP51 in transfected cells. Cells were transfected as indicated with either control empty vector (CTRL), HA-PTPIP51+CTRL, or HA-PTPIP51+ either EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown and no immunoprecipitating VAPB signals were obtained in the absence of HA-PTPIP51. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals.


( A ) overexpression of FUS does not affect expression of VAPB , PTPIP51 or mitofusin - 2 . Immunoblots of spinal cord proteins from three 10 week old FUS transgenic mice and three age - matched littermates are shown ; FUS was detected via its HA - tag . Tubulin was used as a loading control .

(A) overexpression of FUS does not affect expression of VAPB, PTPIP51 or mitofusin-2. Immunoblots of spinal cord proteins from three 10 week old FUS transgenic mice and three age-matched littermates are shown; FUS was detected via its HA-tag. Tubulin was used as a loading control.


( B ) representative electron microscopy of ER - mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice and their non - transgenic littermates ; arrowheads with loops show regions of association . Scale bar is 200 nm . Bar chart shows % of mitochondrial surface closely apposed to ER in the two samples . Data were analysed by unpaired t - test . N = 67 - 88 cells and 438 - 749 mitochondria , error bars are s . e . m . ; * * * p < 0 . 001 .

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(B) representative electron microscopy of ER-mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice and their non-transgenic littermates; arrowheads with loops show regions of association. Scale bar is 200 nm. Bar chart shows % of mitochondrial surface closely apposed to ER in the two samples. Data were analysed by unpaired t-test. N=67-88 cells and 438-749 mitochondria, error bars are s.e.m.; ***p<0.001.


( C ) ER - mitochondria associations and the VAPB - PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice . Representative images of proximity ligation signals in 11 week old FUS and non - transgenic ( NTg ) littermate mice . Data were analysed by unpaired t - test ; N = 40 cells from 3 FUS and 3 non - transgenic littermates ( age 10 weeks ) .

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(C) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice. Representative images of proximity ligation signals in 11 week old FUS and non-transgenic (NTg) littermate mice. Data were analysed by unpaired t-test; N=40 cells from 3 FUS and 3 non-transgenic littermates (age 10 weeks).


( A and B ) FUS disrupts Ca2 + homeostasis . HEK293 cells were transfected with M3R and either control vector ( CTRL ) , FUS , FUSR521C or FUSR518K as indicated . Release of ERCa2 + was induced by treatment of cells with OxoM . ( A ) shows cytosolicCa2 + levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right . Fluo4 fluorescence shows a transient increase in cytosolicCa2 + levels upon OxoM treatment but compared to control , wild - type and mutant FUS all increase peak cytosolicCa2 + levels . ( B ) shows mitochondrialCa2 + levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right . Data were analysed by one - way ANOVA and Tukey ' s post hoc test . ( A ) N = 49 - 52 cells from 3 experiments ; ( B ) N = 50 - 52 cells from 5 experiments , error bars are s . e . m . ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O 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 B-CONTROLLED_VAR 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 B-MEASURED_VAR O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) FUS disrupts Ca2+ homeostasis. HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K as indicated. Release of ERCa2+ was induced by treatment of cells with OxoM. (A) shows cytosolicCa2+ levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right. Fluo4 fluorescence shows a transient increase in cytosolicCa2+ levels upon OxoM treatment but compared to control, wild-type and mutant FUS all increase peak cytosolicCa2+ levels. (B) shows mitochondrialCa2+ levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right. Data were analysed by one-way ANOVA and Tukey's post hoc test. (A) N=49-52 cells from 3 experiments; (B) N=50-52 cells from 5 experiments, error bars are s.e.m.; *p<0.05, **p<0.01, ***p<0.001.


( C ) FUS reduces mitochondrial ATP production . ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1 . 03 and either control vector ( CTRL ) , HA - FUS , HA - FUSR521C or HA - FUSR518K . Cells were imaged in time - lapse prior to and after KCN treatment to inhibit oxidative phosphorylation . Representative traces of YFP / CFP ratios are shown for the different samples ; initial YFP / CFP ratios prior to KCN treatment and those after KCN treatment are indicated . The fall in YFP / CFP ratios correlates with ATP produced by oxidative phosphorylation . Bar chart shows relative ATP levels produced by oxidative phosphorylation ( OXPHOS ) in the different samples .

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(C) FUS reduces mitochondrial ATP production. ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1.03 and either control vector (CTRL), HA-FUS, HA-FUSR521C or HA-FUSR518K. Cells were imaged in time-lapse prior to and after KCN treatment to inhibit oxidative phosphorylation. Representative traces of YFP/CFP ratios are shown for the different samples; initial YFP/CFP ratios prior to KCN treatment and those after KCN treatment are indicated. The fall in YFP/CFP ratios correlates with ATP produced by oxidative phosphorylation. Bar chart shows relative ATP levels produced by oxidative phosphorylation (OXPHOS) in the different samples.


( A ) cells were transfected with either control vector ( CTRL ) , HA - FUS , HA - FUSR521C or HA FUSR518K and the samples probed on immunoblots for GSK - 3 phosphorylated on serine - 9 ( GSK - 3 - S9 ) , total GSK - 3 , FUS ( using FUS antibody ) and tubulin as a loading control . Phosphorylation of GSK - 3 serine - 9 is the principal mechanism for regulating its activity ; serine - 9 phosphorylation inhibits GSK - 3 activity . Bar chart shows relative levels of GSK - 3β serine - 9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK - 3β signals . Data were analysed by one - way ANOVA and Tukey ' s post hoc test . N = 4 , error bars are s . e . m ; * p < 0 . 05 .

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(A) cells were transfected with either control vector (CTRL), HA-FUS, HA-FUSR521C or HA FUSR518K and the samples probed on immunoblots for GSK-3 phosphorylated on serine-9 (GSK-3-S9), total GSK-3, FUS (using FUS antibody) and tubulin as a loading control. Phosphorylation of GSK-3 serine-9 is the principal mechanism for regulating its activity; serine-9 phosphorylation inhibits GSK-3 activity. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way ANOVA and Tukey's post hoc test. N=4, error bars are s.e.m; *p<0.05.


( B ) immunoblots of total and serine - 9 phosphorylated GSK - 3 in spinal cords from three 10 week - old FUS transgenic mice and their non - transgenic littermates . Samples were probed for tubulin as a loading control . Bar chart shows relative levels of GSK - 3β serine - 9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK - 3β signals . Data were analysed by unpaired t - test , error bars are s . e . m . ; * p < 0 . 05 .

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(B) immunoblots of total and serine-9 phosphorylated GSK-3 in spinal cords from three 10 week-old FUS transgenic mice and their non-transgenic littermates. Samples were probed for tubulin as a loading control. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by unpaired t-test, error bars are s.e.m.; *p<0.05.


( C ) cytosolic FUS activates GSK - 3 more potently than wild type FUS . Cells were transfected with EGFP control , EGFP - FUS or EGFP - FUS lacking its C - terminal nuclear localization signal ( FUSC ) . Samples were probed on immunoblots for total GSK - 3 , GSK - 3 phosphorylated on serine - 9 , FUS ( using EGFP antibody ) and tubulin as a loading control . Bar chart shows relative levels of GSK - 3β serine - 9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK - 3β signals . Data were analysed by one - way ANOVA and Tukey ' s post hoc test . N = 3 , error bars are s . e . m . ; * * p < 0 . 01 , * * * p < 0 . 001 .

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(C) cytosolic FUS activates GSK-3 more potently than wild type FUS. Cells were transfected with EGFP control, EGFP-FUS or EGFP-FUS lacking its C-terminal nuclear localization signal (FUSC). Samples were probed on immunoblots for total GSK-3, GSK-3 phosphorylated on serine-9, FUS (using EGFP antibody) and tubulin as a loading control. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way ANOVA and Tukey's post hoc test. N=3, error bars are s.e.m.; **p<0.01, ***p<0.001.


( D ) cytosolic FUS reduces the VAPB - PTPIP51 interaction more potently than wild type FUS . Cells were transfected with PTPIP51 - HA and either EGFP control , EGFP - FUS or EGFP - FUSC . PTPIP51 was immunoprecipitated using the HA tag and bound endogenous VAPB detected by immunoblotting . Both inputs and immunoprecipitations ( IP ) are shown . FUS was detected using EGFP antibody . Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots . VAPB signals were normalized to immunoprecipitated PTPIP51 - HA signals .

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(D) cytosolic FUS reduces the VAPB-PTPIP51 interaction more potently than wild type FUS. Cells were transfected with PTPIP51-HA and either EGFP control, EGFP-FUS or EGFP-FUSC. PTPIP51 was immunoprecipitated using the HA tag and bound endogenous VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown. FUS was detected using EGFP antibody. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals.


( A ) representative electron microscopy of ER - mitochondria associations in NSC34 cells treated with either vehicle or GSK - 3β inhibitors AR - A014418 ( 1 M ) or CT99021 ( 100 nM ) for 16 h . Arrowheads with loops show regions of association ; scale bar = 200 nm . Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples . Data were analysed by one - way ANOVA followed by Tukey ' s multiple comparison test . N = 32 cells and 181 - 210 mitochondria . Error bars are s . e . m . ; * p < 0 . 05 .

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(A) representative electron microscopy of ER-mitochondria associations in NSC34 cells treated with either vehicle or GSK-3β inhibitors AR-A014418 (1 M) or CT99021 (100 nM) for 16 h. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test. N=32 cells and 181-210 mitochondria. Error bars are s.e.m.; *p<0.05.


( B ) VAPB - PTPIP51 proximity ligation assays of NSC34 cells treated with either vehicle 1 M AR - A014418 or 100 nM CT99021 for 16 h . Cells were also stained for nuclei with DAPI . Bar chart shows relative number of proximity ligation assay signals / cell .

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(B) VAPB-PTPIP51 proximity ligation assays of NSC34 cells treated with either vehicle 1 M AR-A014418 or 100 nM CT99021 for 16 h. Cells were also stained for nuclei with DAPI. Bar chart shows relative number of proximity ligation assay signals/cell.


Representative electron microscopy of ER - mitochondria associations in NSC34 cells transfected with EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K and treated with vehicle or 1 mM AR - A014418 for 16 hours . Arrowheads with loops show regions of association ; scale bar = 200 nm . Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples .

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Representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and treated with vehicle or 1 mM AR-A014418 for 16 hours. Arrowheads with loops show regions of association; scale bar=200 nm. Bar charts shows % of the mitochondrial surface closely apposed to ER in the different samples.


HEK293 cells were transfected with M3R and either control vector ( CTRL ) , FUS , FUSR521C or FUSR518K and then treated with vehicle ( DMSO ) or 1 M AR - A014418 for 16 hours as indicated . Release of ER Ca2 + was induced by treatment of cells with OxoM . Representative Rhod2 fluorescence traces showing mitochondrial Ca2 + are shown along with bar chart displaying normalized peak values .

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HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K and then treated with vehicle (DMSO) or 1 M AR-A014418 for 16 hours as indicated. Release of ER Ca2+ was induced by treatment of cells with OxoM. Representative Rhod2 fluorescence traces showing mitochondrial Ca2+ are shown along with bar chart displaying normalized peak values.


A Localisation of transiently expressed GFP - Rab35 in IMCD3 cells after 48h serum starvation and staining for acetylated tubulin ( acetyl . tub . ) and DNA . Insets show higher magnification images of the cilia region . Scale bars , 10 µm .

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A Localisation of transiently expressed GFP-Rab35 in IMCD3 cells after 48h serum starvation and staining for acetylated tubulin (acetyl. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.


B Localisation of transiently expressed GFP - RAB35 in hTERT - RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin ( polyglu . tub . ) and DNA . Insets show higher magnification images of the cilia region . Scale bars , 10 µm .

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B Localisation of transiently expressed GFP-RAB35 in hTERT-RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin (polyglu. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 µm.


C Super resolution ( FV - OSR ) imaging of hTERT - RPE1 stably expressing GFP - RAB35 after 24 h serum starvation and staining for GFP , ARL13B ( cilia membrane ) , IFT88 ( axoneme ) , acetylated tubulin ( acetyl . tub . ; axoneme ) , and γ - tubulin ( γ - tub ; centrosome ) . Top panels show representative images and bottom panel shows line profile plots of fluorescence intensity ( arbitrary units ; a . u . ) in the distal ( dotted line ) and proximal ( dashed line ) cilia regions . Data are mean ± S . E . M . ( n = 5 cilia ) , with the solid line in the line profile plots indicating mean and the dotted lines indicating S . E . M . values . Scale bars , 1 µm .

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C Super resolution (FV-OSR) imaging of hTERT-RPE1 stably expressing GFP-RAB35 after 24 h serum starvation and staining for GFP, ARL13B (cilia membrane), IFT88 (axoneme), acetylated tubulin (acetyl. tub.; axoneme), and γ-tubulin (γ-tub; centrosome). Top panels show representative images and bottom panel shows line profile plots of fluorescence intensity (arbitrary units; a. u.) in the distal (dotted line) and proximal (dashed line) cilia regions. Data are mean ± S.E.M. (n = 5 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating S.E.M. values. Scale bars, 1 µm.


D Representative FV - OSR images of 24 h serum - starved hTERT - RPE1 stably expressing GFP - RAB35 and stained for GFP , ARL13B and acetylated tubulin . Right panels show a cilium with proximal enrichment of GFP - RAB35 and left panels a cilium with even GFP - RAB35 localisation along the full length . Scale bars ; 1 µm .

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D Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, ARL13B and acetylated tubulin. Right panels show a cilium with proximal enrichment of GFP-RAB35 and left panels a cilium with even GFP-RAB35 localisation along the full length. Scale bars; 1 µm.


E Representative FV - OSR images of 24 h serum - starved hTERT - RPE1 stably expressing GFP - RAB35 and stained for GFP , EHD1 and ARL13B . Line profile plots of fluorescence intensity in the proximal cilia region ( dashed line ) are shown to the right . Data are mean ± S . E . M . ( n = 6 cilia ) , with the solid line in the line profile plots indicating mean and the dotted lines indicating S . E . M . values . Scale bars ; 1 µm .

E Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, EHD1 and ARL13B. Line profile plots of fluorescence intensity in the proximal cilia region (dashed line) are shown to the right. Data are mean ± S.E.M. (n = 6 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating S.E.M. values. Scale bars; 1 µm.


A Immunoblot analysis of RAB35 depletion in hTERT - RPE1 cells transfected with two independent siRNAs targeting RAB35 ( RAB35 - 1 , RAB35 - 2 ) or a non - targeting siRNA control ( Neg ) . 24 h after transfection , cells were serum - starved for further 48 h . β - tubulin served as a loading control .

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A Immunoblot analysis of RAB35 depletion in hTERT-RPE1 cells transfected with two independent siRNAs targeting RAB35 (RAB35-1, RAB35-2) or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. β-tubulin served as a loading control.


B Quantification of ciliation of hTERT - RPE1 cells treated and using acetylated tubulin staining as a cilia marker . Data are mean ± S . E . M . of 4 independent experiments ( n ≥ 50 cilia per experimental condition ) .

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B Quantification of ciliation of hTERT-RPE1 cells treated and using acetylated tubulin staining as a cilia marker. Data are mean ± S.E.M. of 4 independent experiments (n ≥ 50 cilia per experimental condition).


C , D hTERT - RPE1 cells and hTERT - RPE1 cells stably expressing siRNA - resistant GFP - RAB35 were treated and stained for GFP , acetylated tubulin ( acetyl . tub . ) and DNA . Representative images in ( C ) of cells treated with Neg or RAB35 siRNA . Regions within white boxes shown at higher magnifications to the right . Scale bars , 10 µm . Cilia length quantifications in ( D ) are shown as box - and - whisker plots . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment of three is shown ( n ≥ 50 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . : non - significant ; P - values : hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P < 0 . 0001 , hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P < 0 . 0001 , hTERT - RPE1 - RAB35 - 1 vs . GFP - RAB35 - RAB35 - 1 P = 0 . 0249 , hTERT - RPE1 - RAB35 - 2 vs . GFP - RAB35 - RAB35 - 2 P = 0 . 0056 )

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C, D hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 were treated and stained for GFP, acetylated tubulin (acetyl. tub.) and DNA. Representative images in (C) of cells treated with Neg or RAB35 siRNA. Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 µm. Cilia length quantifications in (D) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0249, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0056)


E Immunoblot analysis of Rab35 depletion in IMCD3 cells transfected with pool of siRNAs targeting mouse Rab35 or a non - targeting siRNA control ( Neg ) . 24 h after transfection , cells were serum - starved for further 48 h . GAPDH served as a loading control .

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E Immunoblot analysis of Rab35 depletion in IMCD3 cells transfected with pool of siRNAs targeting mouse Rab35 or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. GAPDH served as a loading control.


F Quantification of ciliation of IMCD3 cells treated and using acetylated tubulin staining as a cilia marker . Data are mean ± S . E . M . of 3 independent experiments ( n ≥ 50 cilia per experimental condition ) .

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F Quantification of ciliation of IMCD3 cells treated and using acetylated tubulin staining as a cilia marker. Data are mean ± S.E.M. of 3 independent experiments (n ≥ 50 cilia per experimental condition).


G - H Immunofluorescence of IMCD3 representative images treated cells are stained for acetylated tubulin ( acetyl . tub . ) and DNA . Cilia length quantification in ( H ) is shown as box - and - whisker plots and is the result of 3 independent experiments ( n ≥ 50 cilia per experimental condition ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . Statistical significance according to unpaired t - test with Mann - Whitney test ( * * * P = 0 . 0002 )

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G-H Immunofluorescence of IMCD3 representative images treated cells are stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (H) is shown as box-and-whisker plots and is the result of 3 independent experiments (n ≥ 50 cilia per experimental condition). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to unpaired t-test with Mann-Whitney test (*** P = 0.0002)


I Immunoblot analysis of NIH3T3 wild type ( WT ) and Rab35 knockout ( KO ) cell lines . β - tubulin served as a loading control .

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I Immunoblot analysis of NIH3T3 wild type (WT) and Rab35 knockout (KO) cell lines. β-tubulin served as a loading control.


NIH3T3 WT and Rab35 KO cell lines were serum - starved for 24h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Quantification of ciliation in ( J ) . Data are mean ± S . E . M . of 3 independent experiments ( n ≥ 100 cilia per experimental condition ) .

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NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Quantification of ciliation in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 100 cilia per experimental condition).


NIH3T3 WT and Rab35 KO cell lines were serum - starved for 24h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Representative images in ( K ) . Regions within white boxes shown at higher magnifications at the bottom . Scale bars , 10 µm . Cilia length quantifications in ( L ) are shown as box - and - whisker plots . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment of three is shown ( n ≥ 100 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * * * P < 0 . 0001 ) .

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NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images in (K). Regions within white boxes shown at higher magnifications at the bottom. Scale bars, 10 µm. Cilia length quantifications in (L) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 100 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (**** P < 0.0001).


A Representative images of hTERT - RPE1 cells transiently expressing wild type ( WT ) , GDP - bound ( S22N ) or GTP - bound ( Q67L ) GFP - tagged RAB35 . Cells were serum - starved for 24 h and stained for polyglutamylated tubulin ( polyglu . tub . ) and DNA . Higher magnification images of the cilia region shown in smaller panels . Scale bars ; 10 µm .

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A Representative images of hTERT-RPE1 cells transiently expressing wild type (WT), GDP-bound (S22N) or GTP-bound (Q67L) GFP-tagged RAB35. Cells were serum-starved for 24 h and stained for polyglutamylated tubulin (polyglu. tub.) and DNA. Higher magnification images of the cilia region shown in smaller panels. Scale bars; 10 µm.


Quantification of GFP - RAB35 ciliary localisation ( B ) in hTERT - RPE1 cells transiently expressing indicated GFP - RAB35 constructs or GFP . Data are mean ± S . E . M . of 3 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * * P < 0 . 01 ; P - values : ( B ) P = 0 .

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Quantification of GFP-RAB35 ciliary localisation (B) in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test ( ** P < 0.01; P-values: (B) P = 0.0015


Quantification of ciliation ( C ) in hTERT - RPE1 cells transiently expressing indicated GFP - RAB35 constructs or GFP . Data are mean ± S . E . M . of 3 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * * P < 0 . 01 ; P - values : ( C ) P = 0 . 0062 ) .

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Quantification of ciliation (C) in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data are mean ± S.E.M. of 3 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test ( ** P < 0.01; P-values: (C) P = 0.0062).


Quantification of ciliary length in hTERT - RPE1 cells transiently expressing indicated GFP - RAB35 constructs or GFP . Cilia length quantification in ( D ) is shown as box - and - whisker plots . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n ≥ 50 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * P < 0 . 05 , * * P < 0 . 01 ; P - values : GFP vs . GFP - RAB35 - S22N P = 0 . 0015 , GFP vs . GFP - RAB35 - Q67L P = 0 . 0158 )

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Quantification of ciliary length in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Cilia length quantification in (D) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (* P < 0.05, ** P < 0.01; P-values: GFP vs. GFP-RAB35-S22N P = 0.0015, GFP vs. GFP-RAB35-Q67L P = 0.0158)


Analysis of cilia length in hTERT - RPE1 cells depleted of GEF DENND1B ) and GAP ( TBC1D10A regulators of RAB35 . Cells transfected with indicated siRNAs were serum - starved for 48 h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Representative images are shown in ( E ) . Regions within white boxes shown at higher magnifications to the right . Scale bars ; 10 µm .

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Analysis of cilia length in hTERT-RPE1 cells depleted of GEF DENND1B) and GAP (TBC1D10A regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Representative images are shown in (E). Regions within white boxes shown at higher magnifications to the right. Scale bars; 10 µm.


Analysis of cilia length in hTERT - RPE1 cells depleted of GEF ( DENND1A , DENND1B ) and GAP ( TBC1D10A , TBC1D10B ) regulators of RAB35 . Cells transfected with indicated siRNAs were serum - starved for 48 h and stained for acetylated tubulin ( acetyl . tub . ) and DNA . Cilia length quantification in ( F ) is shown as box - and - whisker plots . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n ≥ 50 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * P < 0 . 01 , * * * P < 0 . 001 ; P - values : Neg vs . DENND1B P = 0 . 0033 , Neg vs . TBC1D10A P = 0 . 0008 )

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Analysis of cilia length in hTERT-RPE1 cells depleted of GEF (DENND1A, DENND1B) and GAP (TBC1D10A, TBC1D10B) regulators of RAB35. Cells transfected with indicated siRNAs were serum-starved for 48 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (F) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001; P-values: Neg vs. DENND1B P = 0.0033, Neg vs. TBC1D10A P = 0.0008)


G Quantification of ciliary localisation of GFP - RAB35 in hTERT - RPE1 cells stably expressing GFP - RAB35 treated Data are mean ± S . E . M . of 5 independent experiments . Statistical significance according to ANOVA followed by Bonferroni post - hoc test ( * P < 0 . 05 , * * * P < 0 . 001 ; P - values : Neg vs . DENND1B P < 0 . 0001 , Neg vs . TBC1D10A P = 0 . 0322 ) .

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G Quantification of ciliary localisation of GFP-RAB35 in hTERT-RPE1 cells stably expressing GFP-RAB35 treated Data are mean ± S.E.M. of 5 independent experiments. Statistical significance according to ANOVA followed by Bonferroni post-hoc test (* P < 0.05, *** P < 0.001; P-values: Neg vs. DENND1B P < 0.0001, Neg vs. TBC1D10A P = 0.0322).


H - J Localisation of transiently expressed GFP - DENND1B ( H ) or GFP - TBC1D10A ( I ) in hTERT - RPE1 cells after 24 h serum starvation and staining with indicated antibodies . Higher magnification images of the cilia region are shown in smaller panels . Scale bars ; 5 µm . Quantification of localisation to ciliary base or along axoneme in ( J ) . Data are mean ± S . E . M . of 3 independent experiments ( n ≥ 30 cilia per experimental condition ) .

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H-J Localisation of transiently expressed GFP-DENND1B (H) or GFP-TBC1D10A (I) in hTERT-RPE1 cells after 24 h serum starvation and staining with indicated antibodies. Higher magnification images of the cilia region are shown in smaller panels. Scale bars; 5 µm. Quantification of localisation to ciliary base or along axoneme in (J). Data are mean ± S.E.M. of 3 independent experiments (n ≥ 30 cilia per experimental condition).


A Immunostaining of representative images of KV embryos at 8 somite stage in which mRNA encoding for mCherry or mCherry - Rab35 WT was injected . Staining of acetylated tubulin was used as cilia marker . Inset shows a higher magnification image of the cilia region , displaying colocalization of mCherry - Rab35 and acetylated tubulin . Scale bar , 10 µm .

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A Immunostaining of representative images of KV embryos at 8 somite stage in which mRNA encoding for mCherry or mCherry-Rab35 WT was injected. Staining of acetylated tubulin was used as cilia marker. Inset shows a higher magnification image of the cilia region, displaying colocalization of mCherry-Rab35 and acetylated tubulin. Scale bar, 10 µm.


B , C Immunoblot analysis with anti - Rab35 and anti - GAPDH antibodies of 40 µg of total lysates of zebrafish embryos injected with Rab35 morpholino ( MO ) at 8 somite stage , compared with non - injected embryos . The plot in ( C ) shows Rab35 protein level in non - injected and Rab35 morphants , normalized to GAPDH protein level . Data are mean ± S . E . M . of 3 independent experiments ( n > 50 embryos per condition ) .

B,C Immunoblot analysis with anti-Rab35 and anti-GAPDH antibodies of 40 µg of total lysates of zebrafish embryos injected with Rab35 morpholino (MO) at 8 somite stage, compared with non-injected embryos. The plot in (C) shows Rab35 protein level in non-injected and Rab35 morphants, normalized to GAPDH protein level. Data are mean ± S.E.M. of 3 independent experiments (n > 50 embryos per condition).


D Organ situs was determined by observing the heart position at 30 hours post - fertilisation ( hpf ) , and the liver position at 53 hpf , by in situ hybridisation with a probe for foxa3 , in controls ( Mismatch MO and non - injected [ WT ] ) and Rab35 MO . Situs solitus means left heart and liver ; situs inversus means right heart and liver ; and heterotaxia means any other possible combination of the heart and liver position .

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D Organ situs was determined by observing the heart position at 30 hours post-fertilisation (hpf), and the liver position at 53 hpf, by in situ hybridisation with a probe for foxa3, in controls (Mismatch MO and non-injected [WT]) and Rab35 MO. Situs solitus means left heart and liver; situs inversus means right heart and liver; and heterotaxia means any other possible combination of the heart and liver position.


E Whole - mount in situ hybridization with the dand5 probe at 8 somite stage ( 13 hpf ) in wild type non - injected embryos and mismatch morphants , both presenting right - sided distribution of dand5 , and Rab35 morphants presenting bilateral localization of the dand5 probe . Scale bar , 50 µm . Graph shows quantification of embryos ( % ) with bilateral or right - sided dand5 localisation from both conditions . Statistical significance according to Fisher Exact test ( * * P = 0 . 0002 )

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E Whole-mount in situ hybridization with the dand5 probe at 8 somite stage (13 hpf) in wild type non-injected embryos and mismatch morphants, both presenting right-sided distribution of dand5, and Rab35 morphants presenting bilateral localization of the dand5 probe. Scale bar, 50 µm. Graph shows quantification of embryos (%) with bilateral or right-sided dand5 localisation from both conditions. Statistical significance according to Fisher Exact test (** P = 0.0002)


F - I Immunostaining of representative images of KV embryos at 8 somite stage in which : ( F ) Mismatch or Rab35 MO was injected , as well as a co - injection of Rab35 MO with mRNA encoding mCherry - Rab35 WT [ Rescue ] ; ( H ) mRNA encoding mCherry or mCherry - tagged Rab35 WT , Rab35 - Q67L or Rab35 - S22N were injected . Acetylated tubulin is shown in green as cilia marker . Quantification of KV cilia length in ( F ) and ( H ) is shown respectively in ( G ) and ( I ) as box - and - whisker plots of 3 independent experiments . N ≥ 8 embryos and n > 300 cilia per experimental condition . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * P < 0 . 01 , * * * P < 0 . 001 , ns . not significant ; Mismatch vs Rab35 MO , P < 0 . 001 ; Cherry vs Rab35 - SN , P = 0 . 0063 ; ) . Scale bars , 10 µm .

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F-I Immunostaining of representative images of KV embryos at 8 somite stage in which: (F) Mismatch or Rab35 MO was injected, as well as a co-injection of Rab35 MO with mRNA encoding mCherry-Rab35 WT [Rescue]; (H) mRNA encoding mCherry or mCherry-tagged Rab35 WT, Rab35-Q67L or Rab35-S22N were injected. Acetylated tubulin is shown in green as cilia marker. Quantification of KV cilia length in (F) and (H) is shown respectively in (G) and (I) as box-and-whisker plots of 3 independent experiments. N ≥ 8 embryos and n > 300 cilia per experimental condition. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001, ns. not significant; Mismatch vs Rab35 MO, P < 0.001; Cherry vs Rab35-SN, P = 0.0063; ). Scale bars, 10 µm.


A , B hTERT - RPE1 cells and hTERT - RPE1 cells stably expressing siRNA - resistant GFP - RAB35 transfected with non - targeting siRNA control ( Neg ) or Rab35 siRNA , were serum - starved for 48 h and stained for ARL13B , GFP , polyglutamylated tubulin ( polyglu . tub . ) and DNA . Representative images are shown in ( A ) . Regions within white boxes shown at higher magnifications to the right . Scale bars ; 10 µm . ( B ) Box - and - whisker plots show quantification of ciliary ARL13B intensity in arbitrary units ( a . u . ) . Horizontal lines show 25 , 50 and 75th percentiles ; whiskers extend to minimum and maximum values . One representative experiment out of three is shown ( n > 25 cilia per experimental condition ) . Statistical significance according to Kruskal - Wallis followed by Dunn ' s post - hoc test ( * * P < 0 . 01 , * * * P < 0 . 001 , n . s . : non - significant ; P - values : hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P < 0 . 0001 , hTERT - RPE1 - Neg vs . hTERT - RPE1 - RAB35 - 1 P = 0 . 0001 , hTERT - RPE1 - RAB35 - 1 vs . GFP - RAB35 - RAB35 - 1 P = 0 . 0014 , hTERT - RPE1 - RAB35 - 2 vs . GFP - RAB35 - RAB35 - 2 P = 0 . 0081 ) .

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A, B hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 transfected with non-targeting siRNA control (Neg) or Rab35 siRNA, were serum-starved for 48 h and stained for ARL13B, GFP, polyglutamylated tubulin (polyglu. tub.) and DNA. Representative images are shown in (A). Regions within white boxes shown at higher magnifications to the right. Scale bars; 10 µm. (B) Box-and-whisker plots show quantification of ciliary ARL13B intensity in arbitrary units (a. u.). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n > 25 cilia per experimental condition). Statistical significance according to Kruskal-Wallis followed by Dunn's post-hoc test (** P < 0.01, *** P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P = 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0014, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0081).


C Immunoblot analysis of total ARL13B protein levels in control and RAB35 - depleted cells . β - tubulin served as a loading control . The graph shows ARL13B protein levels normalized ( norm . ) to β - tubulin . Data are mean ± S . E . M . of 4 independent experiments .

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C Immunoblot analysis of total ARL13B protein levels in control and RAB35-depleted cells. β-tubulin served as a loading control. The graph shows ARL13B protein levels normalized (norm.) to β-tubulin. Data are mean ± S.E.M. of 4 independent experiments.