Figure 2B Body weight of Uqcrh - / - mice and wild - type littermates at 1 - 2 weeks ( n = 5 to 10 ) , during the post - weaning period from 5 - 6 weeks of age ( n = 3 to 7 ) and weekly from 6 to 10 weeks ( n = 13 to 66 ) , data are shown as mean ± SD . * * * * p < 0 . 0001 , 2 - Way ANOVA , from 7 . 5wk of age for Uqcrh - / - males and females versus sex - matched controls . Data are shown as single data points and median , or categorical data , males and females pooled , since genotype - related effects were comparable for both sexes from different cohorts . * p < 0 . 05 , * * * p < 0 . 001 , Wilcoxon rank - sum test . C , D Clinical chemistry plasma concentrations of parameters related to acid - base balance ( Lactate and Ammonia ) and metabolic state ( Glucose ) n = 15 Uqcrh - / - and 16 WT pups 1 - 2 - week - old ( C ) ; n = 15 f , 15 m Uqcrh - / - and 15 f , 15 m WT mice aged 8 - 9 weeks ( D ) ; sex not differentiated . Data shown as scatter dot plots with line at median . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , Wilcoxon rank sum test . F Measures related to behaviour and neurological function , data generated by the SHIRPA ( 9 weeks old , F ) , n = 7f , 8m Uqcrh - / - and 8f , 9m WT animals . Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating ( 10 weeks , E right panel ) , n = 7 f / 5 m Uqcrh - / - and 8 f / 9 m WT mice . Sex is not differentiated . Data are shown as scatter dot plots with a line representing the median . * * * p < 0 . 001 , * * * * p < 0 . 0001 , Wilcoxon rank - sum test . G Parameters related to heart function generated by Echocardiography ( ECHO ) and Electrocardiography ( ECG ) , n = 15 f , 15 m Uqcrh - / - and 15 f , 15 m WT mice , 6 weeks old .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B Body weight of Uqcrh -/- mice and wild-type littermates at 1-2 weeks (n = 5 to 10), during the post-weaning period from 5-6 weeks of age (n = 3 to 7) and weekly from 6 to 10 weeks (n = 13 to 66), data are shown as mean ± SD. **** p < 0.0001, 2-Way ANOVA, from 7.5wk of age for Uqcrh-/- males and females versus sex-matched controls. Data are shown as single data points and median, or categorical data, males and females pooled, since genotype-related effects were comparable for both sexes from different cohorts. * p < 0.05, *** p <0.001, Wilcoxon rank-sum test.C, D Clinical chemistry plasma concentrations of parameters related to acid-base balance (Lactate and Ammonia) and metabolic state (Glucose) n = 15 Uqcrh-/- and 16 WT pups 1-2-week-old (C); n = 15 f, 15 m Uqcrh-/- and 15 f,15 m WT mice aged 8-9 weeks (D); sex not differentiated. Data shown as scatter dot plots with line at median. * p < 0.05, ** p < 0.01, **** p < 0.0001, Wilcoxon rank sum test.F Measures related to behaviour and neurological function, data generated by the SHIRPA (9 weeks old, F), n = 7f, 8m Uqcrh-/- and 8f, 9m WT animals. Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating (10 weeks, E right panel), n = 7 f/5 m Uqcrh-/- and 8 f/ 9 m WT mice. Sex is not differentiated. Data are shown as scatter dot plots with a line representing the median. *** p < 0.001, **** p < 0.0001, Wilcoxon rank-sum test.G Parameters related to heart function generated by Echocardiography (ECHO) and Electrocardiography (ECG), n = 15 f, 15 m Uqcrh-/- and 15 f, 15 m WT mice, 6 weeks old.


Figure 3A Respiratory chain enzyme activities from human control ( green ) and patient ( red ) fibroblasts . Mean activities of controls ( n = 8 ) are set to 100 % and error bars represent 1 standard deviation . Data are normalised to citrate synthase ( CS ) activity . B Respiratory chain enzyme activities in heart , brain and liver , of wild - type and Uqcrh - / - mice . Data are normalised to citrate synthase ( CS ) activity . Values are given as mean ± SD , n = 4 WT , 5 Uqcrh - / - ( Heart ) , 3 WT , 3 Uqcrh - / - ( Brain ) , 1 WT and 2 Uqcrh - / - ( Liver ) . * p < 0 . 05 , * * * * p < 0 . 0001 , 2 - way ANOVA ( Multiple comparison ) . C Western blot analysis of OXPHOS components in the patient ( P ) and control ( n = 2 , C1 - 2 ) fibroblasts . D BN - PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts ( n = 2 , C1 - 2 ) solubilised with DDM . Immunoblotting was performed using antibodies to a subunit of each complex ( CI [ NDUFB8 ] , CII [ SDHA ] , CIII [ UQCRC2 ] , CIV [ MT - CO1 ] , and CV [ ATP5A ] ) F Immunohistochemical staining of UQCRC2 was performed on liver , heart and kidney derived from wild - type and Uqcrh - / - mice at 9 weeks of age . Staining of VDAC1 / porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1 / porin and UQCRC2 . Data are given as mean ± SEM . * p < 0 . 05 ; n = 3 , Student ' s t - test ( unpaired samples ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts. Mean activities of controls (n = 8) are set to 100% and error bars represent 1 standard deviation. Data are normalised to citrate synthase (CS) activity. B Respiratory chain enzyme activities in heart, brain and liver, of wild-type and Uqcrh-/- mice. Data are normalised to citrate synthase (CS) activity. Values are given as mean ± SD, n = 4 WT, 5 Uqcrh-/- (Heart), 3 WT, 3 Uqcrh-/- (Brain), 1 WT and 2 Uqcrh-/- (Liver). * p < 0.05, **** p < 0.0001, 2-way ANOVA (Multiple comparison). C Western blot analysis of OXPHOS components in the patient (P) and control (n = 2, C1-2) fibroblasts. D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]) F Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh-/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. * p < 0.05; n = 3, Student's t-test (unpaired samples).


Figure 4A Western blot analysis of control ( n = 2 , C1 - 2 ) and patient fibroblasts transduced with a lentiviral vector ( pLVX ) containing wild - type UQCRH . Expression of wild - type UQCRH was induced using various concentrations of doxycycline ( dox ) up to 20 ng / ml for 72 hours . B BN - PAGE analysis of control ( n = 2 , C1 - 2 ) and patient fibroblasts transduced with a lentiviral vector ( pLVX ) containing wild - type UQCRH . Transduced fibroblasts were either uninduced or induced with 20 ng / ml doxycycline ( dox ) for 72 hours . C Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts . D Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in patient fibroblasts . E Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts transduced with wild - type UQCRH . F Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in patient fibroblasts transduced with wild - type ( WT ) UQCRH . G Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in control ( NHDF ) fibroblasts transduced with the pseudogene UQCRHL . H Immunofluorescence for UQCRC2 ( green ) , VDAC1 / porin ( red ) and nuclei ( DAPI , blue ) , in patient fibroblasts transduced with the pseudogene UQCRHL . I Graph indicates the staining intensity of UQCRC2 in control ( NHDF ) fibroblasts ( green ) and patient fibroblasts ( red ) , those respective cell lines transduced ( T ) with wild - type UQCRH ( diagonal lines ) and those cell lines transduced with the pseudogene UQCRHL ( vertical lines ) measured using Image J , Data are given as mean ± SEM . One - way ANOVA ( Kruskal - Wallis test ) , * p < 0 . 05 ; n = 3 macroscopic field ( 10 - 14 cells for macroscopic field were measured ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Western blot analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Expression of wild-type UQCRH was induced using various concentrations of doxycycline (dox) up to 20 ng/ml for 72 hours. B BN-PAGE analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Transduced fibroblasts were either uninduced or induced with 20 ng/ml doxycycline (dox) for 72 hours. C Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts. D Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts. E Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with wild-type UQCRH. F Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with wild-type (WT) UQCRH. G Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with the pseudogene UQCRHL. H Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with the pseudogene UQCRHL. I Graph indicates the staining intensity of UQCRC2 in control (NHDF) fibroblasts (green) and patient fibroblasts (red), those respective cell lines transduced (T) with wild-type UQCRH (diagonal lines) and those cell lines transduced with the pseudogene UQCRHL (vertical lines) measured using Image J, Data are given as mean ± SEM. One-way ANOVA (Kruskal-Wallis test), * p < 0.05; n = 3 macroscopic field (10-14 cells for macroscopic field were measured).


Figure 5Mitochondrial membranes from fibroblasts of control ( WT ) and patient fibroblasts ( i , immortalised ) were solubilised with digitonin and separated on native gradient gels . A BN - PAGE gel stained with Coomassie showing molecular weight marker ( BHM ) and samples from control and patient ( primary fibroblasts , ( I ) = immortalised fibroblasts ) B BN - PAGE gel stained with NADH : NTB reductase activity stain showing molecular weight marker ( BHM ) and samples from control and patient ( primary fibroblasts , ( I ) = immortalised fibroblasts ) C Further complexome analysis of the BN - PAGE gels shown in A and B . Each native lane was cut into even fractions from high to low molecular mass . Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry . Shown here are average IBAQ values of all identified subunits from complexes III , I and IV D Profile of complex III ( averaged IBAQ values ) in the patient ( dashed line ) and control ( solid line ) fibroblasts . E Profile of complexes III ( red ) , I ( yellow ) and IV ( green ) to visualise and assess supercomplex composition and stability in control ( solid lines ) and patient ( dashed lines ) fibroblasts . heart tissue from wild - type ( WT ) and Uqcrh - / - mice were solubilised with digitonin and separated on native gradient gels . F BN - PAGE gel stained with Coomassie ( left ) and NADH : NTB reductase activity stain ( right ) showing molecular weight marker ( BHM ) and heart tissue samples from wild - type ( WT ) and Uqcrh - / - mice . G BN - PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild - type ( WT ) and Uqcrh - / - mice . H Further complexome analysis of the BN - PAGE gels shown in F , shown here are average IBAQ values of subunits from complexes III , I and IV I Profile of complex III ( averaged IBAQ values ) in wild - type ( + / + , solid line ) and Uqcrh - / - ( - / - , dashed line ) heart tissue . J Profile of complexes III ( red ) , I ( yellow ) and IV ( green ) to visualise and assess supercomplex composition and stability in wild - type ( + / + , solid line ) and Uqcrh - / - ( - / - , dashed line ) heart tissue .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Mitochondrial membranes from fibroblasts of control (WT) and patient fibroblasts (i, immortalised) were solubilised with digitonin and separated on native gradient gels. A BN-PAGE gel stained with Coomassie showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) B BN-PAGE gel stained with NADH:NTB reductase activity stain showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) C Further complexome analysis of the BN-PAGE gels shown in A and B. Each native lane was cut into even fractions from high to low molecular mass. Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry. Shown here are average IBAQ values of all identified subunits from complexes III, I and IV D Profile of complex III (averaged IBAQ values) in the patient (dashed line) and control (solid line) fibroblasts. E Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in control (solid lines) and patient (dashed lines) fibroblasts. heart tissue from wild-type (WT) and Uqcrh-/- mice were solubilised with digitonin and separated on native gradient gels. F BN-PAGE gel stained with Coomassie (left) and NADH:NTB reductase activity stain (right) showing molecular weight marker (BHM) and heart tissue samples from wild-type (WT) and Uqcrh-/- mice. G BN-PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild-type (WT) and Uqcrh-/- mice. H Further complexome analysis of the BN-PAGE gels shown in F, shown here are average IBAQ values of subunits from complexes III, I and IV I Profile of complex III (averaged IBAQ values) in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue. J Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue.


Figure 1 ( A ) Heatmap of differentially enriched metabolites in heart , liver , skeletal muscle and serum of young ( Y ) and aged ( A ) killifish ( n = 4 ) . ( B - C ) Ageing leads to significant cardiac - specific accumulation of sphinganine . ( D ) Targeted lipidomics confirm age - dependent accumulation of sphinganine in killifish ( n = 4 ; per condition ) , zebrafish ( n = 4 , per condition ) , mouse ( n = 4 for young and n = 6 for aged samples ) and human hearts ( n = 1 for young and 2 for aged ) . In the case of human heart tissue , statistical significance was computed from technical replicates . ( E ) Heatmap of differentially expressed transcripts ( Log2 fold change + / - 1 , adjusted p - value cut off < 0 . 05 ) between young and aged killifish hearts ( n = 3 ) . Significantly enriched relevant GO categories depicted as bar graph . ( F ) Transcriptional drift variance of genes involved in genome integrity , epigenetic regulation and sphingolipid metabolism . ( G ) γH2A . X staining of young , mid - aged and aged killifish hearts show signs of DNA damage , increasing with age ( n = 4 , # of sections per animal = 10 alternate sections , 10 µm thickness ) . ( H ) Quantification of γH2A . X + cardiomyocyte nuclei . ( I ) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays . Representative images of single nuclei from young and old hearts along with comet length on the right ( n = 4 ) . ( J ) γH2A . X staining of young and aged human ventricles show signs of DNA damage accumulating with age ( n = 2 , # of sections per sample = 5 alternate sections , 10 µm thickness ) . ( K ) Quantification of γH2A . X + cardiomyocyte nuclei is on the right . Scale bar = 50 µm .

O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Heatmap of differentially enriched metabolites in heart, liver, skeletal muscle and serum of young (Y) and aged (A) killifish (n = 4).(B-C) Ageing leads to significant cardiac-specific accumulation of sphinganine.(D) Targeted lipidomics confirm age-dependent accumulation of sphinganine in killifish (n = 4; per condition), zebrafish (n = 4, per condition), mouse (n = 4 for young and n=6 for aged samples) and human hearts (n = 1 for young and 2 for aged). In the case of human heart tissue, statistical significance was computed from technical replicates.(E) Heatmap of differentially expressed transcripts (Log2 fold change +/- 1, adjusted p-value cut off <0.05) between young and aged killifish hearts (n = 3). Significantly enriched relevant GO categories depicted as bar graph.(F) Transcriptional drift variance of genes involved in genome integrity, epigenetic regulation and sphingolipid metabolism.(G) γH2A.X staining of young, mid-aged and aged killifish hearts show signs of DNA damage, increasing with age (n = 4, # of sections per animal = 10 alternate sections, 10 µm thickness).(H) Quantification of γH2A.X+ cardiomyocyte nuclei.(I) The aged killifish heart shows significant signs of genome instability shown here by neutral comet assays. Representative images of single nuclei from young and old hearts along with comet length on the right (n = 4).(J) γH2A.X staining of young and aged human ventricles show signs of DNA damage accumulating with age (n = 2, # of sections per sample = 5 alternate sections, 10 µm thickness).(K) Quantification of γH2A.X+ cardiomyocyte nuclei is on the right. Scale bar = 50 µm.


Figure 2Sphinganine induces DNA damage in human cardiomyocytes depicted here ( A - C ) by γH2A . X + nuclei in cardiac - specific ACTN2 + cells ( n = 3 biological replicates ; # of cells quantified = 147 per condition ) Sphinganine induces DNA damage in human cardiomyocytes depicted here ( D ) by neutral comet assay of DHS treated hCMs . ( E ) Quantification of comet length in the indicated conditions . Elevated DHS levels lead to ageing signatures in hCMs shown here by ( F - G ) SA - β - GAL activity , Respective graphs depict the quantification of each of the ageing signatures are on the right . Elevated DHS levels lead to ageing signatures in hCMs shown here by ( H ) p21 activation ( # cells quantified per replicate = 150 - 200 ) , ( I ) HP1 - α + nuclear foci ( # of cells quantified per replicate = 120 - 200 ) and ( J ) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining ( # of cells quantified per replicate = 100 - 150 per condition ) . Respective graphs depict the quantification of each of the ageing signatures are on the right . High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs ( L ) and ( M ) in aged human hearts . ( N ) Violin plot depicting the distributions of the grey - scale nuclear intensity of the indicated markers . ( When not specified , the experiments were conducted in at least 3 biological replicates ) . Scale bar = 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Sphinganine induces DNA damage in human cardiomyocytes depicted here (A-C) by γH2A.X+ nuclei in cardiac-specific ACTN2+ cells (n = 3 biological replicates; # of cells quantified = 147 per condition)Sphinganine induces DNA damage in human cardiomyocytes depicted here (D) by neutral comet assay of DHS treated hCMs. (E) Quantification of comet length in the indicated conditions.Elevated DHS levels lead to ageing signatures in hCMs shown here by (F-G) SA-β-GAL activity, Respective graphs depict the quantification of each of the ageing signatures are on the right.Elevated DHS levels lead to ageing signatures in hCMs shown here by (H) p21 activation (# cells quantified per replicate = 150-200), (I) HP1-α+ nuclear foci (# of cells quantified per replicate = 120-200) and (J) disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining (# of cells quantified per replicate = 100-150 per condition). Respective graphs depict the quantification of each of the ageing signatures are on the right.High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs (L) and (M) in aged human hearts. (N) Violin plot depicting the distributions of the grey-scale nuclear intensity of the indicated markers. (When not specified, the experiments were conducted in at least 3 biological replicates). Scale bar = 10 µm.


Figure 3 ( A ) Pharmacological inhibition of HDAC1 using romidepsin , MS - 275 and pyroxamide in human cardiomyocytes cause extensive DNA damage , shown here by γH2A . X staining . ( B ) Bar - graphs depict quantification of γH2A . X + hCM nuclei in the indicated conditions . ( E ) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC - activity assay , shown here as bar graph . ( F ) In vitro HDAC activity assay revealed inhibition of nuclear HDAC ' s and purified HDAC1 activity by DHS1P and S1P , shown here as bar graphs . Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMsAssessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications . ( I ) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co - incubation with RNA Pol II inhibitor , triptolide and ( J ) the respective quantifications of γH2A . X + CM nuclei represented as bar graph . Scale bars for A , I = 10 µm and G = 50 µm . ( When not specified , the experiments were conducted in at least 3 biological replicates ) .

O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Pharmacological inhibition of HDAC1 using romidepsin, MS-275 and pyroxamide in human cardiomyocytes cause extensive DNA damage, shown here by γH2A.X staining.(B) Bar-graphs depict quantification of γH2A.X+ hCM nuclei in the indicated conditions.(E) Sphinganine and its derivative DHS1P inhibits class 1 HDACs in the human cardiomyocytes as inferred from the in cellulo HDAC-activity assay, shown here as bar graph.(F) In vitro HDAC activity assay revealed inhibition of nuclear HDAC's and purified HDAC1 activity by DHS1P and S1P, shown here as bar graphs.Assessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMsAssessment of transcription levels measured by EU labeling assay upon treatment with DHS on hCMs and respective quantifications.(I) Representative micrographs of hCMs indicating the rescue of the DHS induced DNA damage by co-incubation with RNA Pol II inhibitor, triptolide and (J) the respective quantifications of γH2A.X+ CM nuclei represented as bar graph. Scale bars for A,I=10 µm and G = 50 µm. (When not specified, the experiments were conducted in at least 3 biological replicates).


Figure 4 ( A - B ) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS - induced DNA damage as shown here by staining for γH2A . X ( green ) in hCMs . ( C ) Bar graphs representing the percentage of γH2A . X + hCMs in the indicated conditions ( # cells quantified per replicate ~ 200 ) . ( D - E ) Treatment of hCMs with ABC294640 , a specific SPHK2 inhibitor prevents DHS - induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining ( ~ 100 cells quantified / condition ) . ( F ) Violin plots depicting the distributions of the grey - scale nuclear intensity of the indicated markers . ( 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 . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-B) SPHK2 inhibition using ABC294640 and SLM6031434 prevents DHS-induced DNA damage as shown here by staining for γH2A.X (green) in hCMs.(C) Bar graphs representing the percentage of γH2A.X+ hCMs in the indicated conditions (# cells quantified per replicate ~200).(D-E) Treatment of hCMs with ABC294640, a specific SPHK2 inhibitor prevents DHS-induced epigenetic aberrations in hCMs visualized here by H3K56ac and H3K27ac staining (~100 cells quantified/ condition).(F) Violin plots depicting the distributions of the grey-scale nuclear intensity of the indicated markers.(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.(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.(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).


Figure 5 ( 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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Figure 5(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.


Figure 6Representative 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 . ( 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 . ( 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 .

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Figure 6Representative 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.(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.(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.


figf1The 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 . ( 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 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1The 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.(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.


figf2 ( 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 ) . ( 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 ) . ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(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).(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).(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.


figf3 ( 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 ) . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(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).(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.(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).


figf4 ( 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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figf4(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.


figf5 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(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.(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.


figf6 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(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.(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.


figf7 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(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.(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).


figf8 ( 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(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.


Figure 1 ( 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 ] ) . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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]).(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 .


Figure 2 ( 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 . ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.(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.


Figure 3 ( 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 . ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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.(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.


Figure 4 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 1 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.


Figure 2 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.


Figure 3 ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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).


Figure 4 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.


Figure 5 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.


Figure 6 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 7Representative 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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Figure 7Representative 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.


Figure 8HEK293 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8HEK293 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.