L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT - qPCR ( E ) results . Data information : NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT-qPCR (E) results. Data information: NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting ( F ) results . Data information : NC : negative control .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting (F) results. Data information: NC: negative control.


L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before RT - qPCR analysis of Ifna4 ( G ) , expression . Data information : HT - DNA : herring testis DNA ; NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of Ifna4 (G), expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before RT - qPCR analysis of IFNb1 ( H ) , and Cxcl10 ( I ) expression . Data information : HT - DNA : herring testis DNA ; NC : negative control . All data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of IFNb1 (H), and Cxcl10 (I) expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


( J ) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before immunoblotting analysis with the indicated antibodies . Data information : HT - DNA : herring testis DNA ; NC : negative control .

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

(J) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. Data information: HT-DNA: herring testis DNA; NC: negative control.


( A ) Survival analysis of Zdhhc18 + / + and Zdhhc18 - / - mice ( n = 8 each , 6 - 8 weeks old ) intranasally injected with VACV ( 5 × 107 PFU per mouse ) and monitored daily for survival for 13 days using Kaplan - Meier curves and a log - rank ( Mantel - Cox ) test . * * * , P ˂ 0 . 001 .

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

(A) Survival analysis of Zdhhc18+/+ and Zdhhc18-/- mice (n = 8 each, 6-8 weeks old) intranasally injected with VACV (5 × 107 PFU per mouse) and monitored daily for survival for 13 days using Kaplan-Meier curves and a log-rank (Mantel-Cox) test. ***, P˂0.001.


( B ) Representative photomicrographs of H & E - stained liver and kidney tissue sections from mice ( Zdhhc18 + / + or Zdhhc18 - / - ) intravenously injected with HSV - 1 - FS ( 5 × 107 PFU per mouse ) or not after 2 days . Scale bars : 100 μm ( liver ) , 200 μm ( kidney ) . Data information : PFU : plaque - forming units ; H & E : hematoxylin - eosin . Data are representative of at least two independent experiments .

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

(B) Representative photomicrographs of H&E-stained liver and kidney tissue sections from mice (Zdhhc18+/+ or Zdhhc18-/-) intravenously injected with HSV-1-FS (5 × 107 PFU per mouse) or not after 2 days. Scale bars: 100 μm (liver), 200 μm (kidney). Data information: PFU: plaque-forming units; H&E: hematoxylin-eosin. Data are representative of at least two independent experiments.


( C BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments .

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

(C BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments.


D ) BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 . 0001 (

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

D) BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001 (D


( E Frequency of GFP + cells among infected cells ( E ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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

(E Frequency of GFP+ cells among infected cells (E). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


F ) Microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) infected with GFP - HSV - 1 ( MOI = 1 ) for 10 h . Scale bar : 200 μm . Data information : Data are representative of at least two independent experiments .

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

F) Microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18-/-) infected with GFP-HSV-1 (MOI=1) for 10 h. Scale bar: 200 μm. Data information: Data are representative of at least two independent experiments.


( G ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before immunoblot analysis .

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

(G) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before immunoblot analysis.


( H and I ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of Cxcl10 ( H ) and Ifnb1 ( I ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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

(H and I) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before RT-qPCR analysis of Cxcl10 (H) and Ifnb1 (I) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


( J ) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA ( cocktail ) for 48 h and then infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of IFNB1 expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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

(J) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA (cocktail) for 48 h and then infected with VACV (1:200) for the indicated times before RT-qPCR analysis of IFNB1 expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs . Passage 6 cells represent the proliferating state , and passages 21 and 34 the senescent state for HUVECs and IMR90 , respectively .

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

Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs. Passage 6 cells represent the proliferating state, and passages 21 and 34 the senescent state for HUVECs and IMR90, respectively.


Super - resolution ( gSTED ) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI . Bar : 2μm .

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

Super-resolution (gSTED) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI. Bar: 2μm.


Representative immunofluorescence images of IMR90 and HUVECs ( left ) showing reduced HMGB1 levels in senescent nuclei ; bean plots quantify this reduction ( right ; N is the number of cells analyzed per each condition / cell type ) . Bars : 5 μm . * : P < 0 . 01 ; Wilcoxon - Mann - Whitney test .

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

Representative immunofluorescence images of IMR90 and HUVECs (left) showing reduced HMGB1 levels in senescent nuclei; bean plots quantify this reduction (right; N is the number of cells analyzed per each condition/cell type). Bars: 5 μm. *: P<0.01; Wilcoxon-Mann-Whitney test.


Genome browser view of HMGB1 ChIP - seq ( normalized to input ; mean from two replicates ) from proliferating HUVEC ( red ) or proliferating / senescent IMR90 ( grey / green ) in the TMEM92 locus .

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

Genome browser view of HMGB1 ChIP-seq (normalized to input; mean from two replicates) from proliferating HUVEC (red) or proliferating/senescent IMR90 (grey/green) in the TMEM92 locus.


Heatmap showing protein ( SASP , from http : / / www . saspatlas . com / ; log fold ratio ) and gene expression levels ( log2FC ) of IMR90 SASP - related genes embedded in high - HMGB1 ( red ) TADs like those in panel C . * * : genes bound by HMGB1 in ChIP - seq data are more than expected by chance ; P > 0 . 001 , hypergeometric test .

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

Heatmap showing protein (SASP, from http://www.saspatlas.com/; log fold ratio)and gene expression levels (log2FC) of IMR90 SASP-related genes embedded in high-HMGB1 (red) TADs like those in panel C. **: genes bound by HMGB1 in ChIP-seq data are more than expected by chance; P>0.001, hypergeometric test.


Immunofluorescence ( top ) , western blot ( middle ) and RT - qPCR analyses ( bottom ; mean fold - change ± S . D . from two biological replicates ) confirm HMGB1 knockdown in IMR90 .

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

Immunofluorescence (top), western blot (middle) and RT-qPCR analyses (bottom; mean fold-change ±S.D. from two biological replicates) confirm HMGB1 knockdown in IMR90.


Representative images of IMR90 overexpressing HMGB1 - GFP , immunostained for p21 and CTCF , and counterstained with DAPI . IMR90 transfected with empty vectors provide a control . Bar : 5 μm .

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

Representative images of IMR90 overexpressing HMGB1-GFP, immunostained for p21 and CTCF, and counterstained with DAPI. IMR90 transfected with empty vectors provide a control. Bar: 5 μm.


Genome browser views showing HMGB1 sCLIP data ( black ) along the ASH1L , CCNL2 and TET2 loci ; input tracks ( grey ) provide background levels . * : significantly - enriched peaks . The consensus motif for HMGB1 binding on RNA is also shown ( bottom right ) .

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

Genome browser views showing HMGB1 sCLIP data (black) along the ASH1L, CCNL2 and TET2 loci; input tracks (grey) provide background levels. *: significantly-enriched peaks. The consensus motif for HMGB1 binding on RNA is also shown (bottom right).


Bar graphs showing mean fold - change of selected mRNAs ( over ± S . D . from two biological replicates ) from ILF3 - ( purple ) , RBMX - ( white ) or PNN - knockdown experiments ( blue ) in proliferating IMR90 . * : significantly different to siRNA controls ; P < 0 . 01 , unpaired two - tailed Student ' s t - test . Dashed line indicates no change in expression .

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

Bar graphs showing mean fold-change of selected mRNAs (over ±S.D. from two biological replicates) from ILF3- (purple), RBMX- (white) or PNN-knockdown experiments (blue) in proliferating IMR90. *: significantly different to siRNA controls; P<0.01, unpaired two-tailed Student's t-test. Dashed line indicates no change in expression.


B . Western blot analysis of RAB6A / A ' and RAB6B protein levels in WT , Emx1 - Cre ; RAB6loxP / loxP ( RAB6A KO ) , RAB6B - / - ( RAB6B KO ) and Emx1 - Cre ; RAB6AloxP / loxP ; RAB6B - / - ( RAB6A / B dKO ) E15 . 5 cortical extracts .

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

B. Western blot analysis of RAB6A/A' and RAB6B protein levels in WT, Emx1-Cre; RAB6loxP/loxP (RAB6A KO), RAB6B-/- (RAB6B KO) and Emx1-Cre; RAB6AloxP/loxP; RAB6B-/- (RAB6A/B dKO) E15.5 cortical extracts.


A . PAX6 staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . B . Percentage of PAX6 + cells located in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 4282 cells from N = 6 brains . RAB6A / B dKO : 1241 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD .

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

A. PAX6 staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. B. Percentage of PAX6+ cells located in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 4282 cells from N=6 brains. RAB6A/B dKO: 1241 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.


C . Phospho - Vimentin ( p - Vim ) staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . D . Percentage p - VIM + cells dividing ectopically , in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 1713 cells from N = 6 brains . RAB6A / B dKO : 506 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD .

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

C. Phospho-Vimentin (p-Vim) staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. D. Percentage p-VIM+ cells dividing ectopically, in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 1713 cells from N=6 brains. RAB6A/B dKO: 506 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.


G . Electroporation of RAB6AloxP / loxP ; RAB6B - / - E14 . 5 embryos with mCherry ( control ) or mCherry + CRE ( RAB6A / B dKO ) and live imaging of delamination events at E17 . 5 . H . Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17 . 5 . mCherry : N = 69 cells from 5 movies . mCherry + CRE : N = 52 cells from 4 movies . Fisher ' s exact test , * p ≤ 0 . 05 . Data information : All error bars indicate SD .

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

G. Electroporation of RAB6AloxP/loxP; RAB6B-/- E14.5 embryos with mCherry (control) or mCherry + CRE (RAB6A/B dKO) and live imaging of delamination events at E17.5. H. Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17.5. mCherry: N=69 cells from 5 movies. mCherry + CRE: N=52 cells from 4 movies. Fisher's exact test, *p ≤ 0.05. Data information: All error bars indicate SD.


A . Localization of the Golgi apparatus ( ManII - GFP ) and the centrosome ( 𝛾 - tubulin ) in E15 . 5 mCherry - electroporated radial glial cell . The Golgi apparatus is localized basally , away from the centrosome . Scale bar = 5µm . B . Average distance between the apical - most part of the Golgi apparatus and the apical surface in aRG cells . N = 224 cells from 3 independent brains .

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

A. Localization of the Golgi apparatus (ManII-GFP) and the centrosome (𝛾-tubulin) in E15.5 mCherry-electroporated radial glial cell. The Golgi apparatus is localized basally, away from the centrosome. Scale bar = 5µm. B. Average distance between the apical-most part of the Golgi apparatus and the apical surface in aRG cells. N=224 cells from 3 independent brains.


E . Live imaging of GFP - RAB6A in aRG cells at E15 . 5 allows tracking of individual RAB6A + vesicles in situ , from the basal Golgi apparatus towards the apical surface . Scale bar = 5µm . Distance = 5µm , time = 30 seconds . F . RAB6A + vesicle directionality in apical processes of aRG cells over one - minute movies . G . Relative time spent by RAB6A + vesicles in apical , basal or static phases . H . Velocity of apically and basally moving RAB6A + vesicles . Data information : ( F , G , H ) N = 388 vesicles from 30 cells . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median .

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

E. Live imaging of GFP-RAB6A in aRG cells at E15.5 allows tracking of individual RAB6A+ vesicles in situ, from the basal Golgi apparatus towards the apical surface. Scale bar = 5µm. Distance = 5µm, time = 30 seconds. F. RAB6A+ vesicle directionality in apical processes of aRG cells over one-minute movies. G. Relative time spent by RAB6A+ vesicles in apical, basal or static phases. H. Velocity of apically and basally moving RAB6A+ vesicles. Data information: (F, G, H) N= 388 vesicles from 30 cells. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.


I Live imaging of GFP - RAB6A in control , dynarrestin - treated and CC1 - p150 - expressing aRG cells at E15 . 5 . Scale bars = 5µm . Distance = 5µm , time = 30 seconds . J . Number of RAB6A + vesicles in the apical process of DMSO and dynarrestin - treated mouse aRG cells . K . Relative time spent by RAB6A + vesicles in apical movement phase , in DMSO and dynarrestin - treated mouse aRG cells . L . Number of RAB6A + vesicles in the apical process of mCherry and CC1 - p150 - expressing aRG cells . M . Relative time spent by RAB6A + vesicles in apical movement phase , in mCherry and CC1 - p150 - expressing aRG cells . DMSO treatment slightly affected RAB6A dynamics , as compared to mCherry control . Data information : ( J , K , L , M ) 216 vesicles from N = 11 cells for DMSO , 145 vesicles from N = 25 cells for dynarrestin , 173 vesicles from N = 17 cells for mCherry control , 71 vesicles from N = 15 cells for CC1 - p150 . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median .

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

I Live imaging of GFP-RAB6A in control, dynarrestin-treated and CC1-p150-expressing aRG cells at E15.5. Scale bars = 5µm. Distance = 5µm, time = 30 seconds. J. Number of RAB6A+ vesicles in the apical process of DMSO and dynarrestin-treated mouse aRG cells. K. Relative time spent by RAB6A+ vesicles in apical movement phase, in DMSO and dynarrestin-treated mouse aRG cells. L. Number of RAB6A+ vesicles in the apical process of mCherry and CC1-p150-expressing aRG cells. M. Relative time spent by RAB6A+ vesicles in apical movement phase, in mCherry and CC1-p150-expressing aRG cells. DMSO treatment slightly affected RAB6A dynamics, as compared to mCherry control. Data information: (J, K, L, M) 216 vesicles from N=11 cells for DMSO, 145 vesicles from N=25 cells for dynarrestin, 173 vesicles from N=17 cells for mCherry control, 71 vesicles from N=15 cells for CC1-p150. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.


C . RUSH assay for CRB3 - GFP in control ( mcherry ) and dynactin - inhibited radial glial cells ( CC1 - p150 - dsRed ) , electroporated at E . 15 . 5 and imaged at E16 . 5 . Scale bar = 5µm . D . CRB3 localization at the apical surface upon release . E . Percentage of cells with 100 % of CRB3 signal at the apical surface upon release . Data information : ( D , E ) mCherry ( control ) : N = 3 brains per timepoint ( 361 cells total ) . CC1 - p150 : N = 3 brains per timepoint ( 2 brains for T0 ) ( 268 cells total ) . Fisher ' s exact test and Benjamini - Hochberg procedure , * * * p ≤ 0 . 001 . All error bars indicate SD .

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

C. RUSH assay for CRB3-GFP in control (mcherry) and dynactin-inhibited radial glial cells (CC1-p150-dsRed), electroporated at E.15.5 and imaged at E16.5. Scale bar = 5µm. D. CRB3 localization at the apical surface upon release. E. Percentage of cells with 100% of CRB3 signal at the apical surface upon release. Data information: (D, E) mCherry (control): N= 3 brains per timepoint (361 cells total). CC1-p150: N=3 brains per timepoint (2 brains for T0) (268 cells total). Fisher's exact test and Benjamini-Hochberg procedure, *** p ≤ 0.001. All error bars indicate SD.


A . PAX6 and phospho - Histone 3 ( p - H3 ) staining in WT and LIS1 KO E12 . 5 brains . Cortices were subdivided into 5 bins of equal size along the radial axis . Scale bar = 25µm . B . Percentage of PH3 + / PAX6 + cells located above the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1192 cells from N = 6 brains . LIS1 KO : 589 cells from N = 3 brains . Data information : ( B , Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

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

A. PAX6 and phospho-Histone 3 (p-H3) staining in WT and LIS1 KO E12.5 brains. Cortices were subdivided into 5 bins of equal size along the radial axis. Scale bar = 25µm. B. Percentage of PH3+/PAX6+ cells located above the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1192 cells from N=6 brains. LIS1 KO: 589 cells from N=3 brains. Data information: (B, Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


C . Phospho - Vimentin ( p - VIM ) staining in WT and LIS1 KO E12 . 5 brains . Scale bar = 25µm . D . Percentage p - VIM + cells dividing ectopically , away from the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1056 cells from N = 6 brains . LIS1 KO : 879 cells from N = 3 brains . Data information : D ) Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

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

C. Phospho-Vimentin (p-VIM) staining in WT and LIS1 KO E12.5 brains. Scale bar = 25µm. D. Percentage p-VIM+ cells dividing ectopically, away from the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1056 cells from N=6 brains. LIS1 KO: 879 cells from N=3 brains. Data information: D) Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


A . CRB3 and PALS1 staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm .

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

A. CRB3 and PALS1 staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.


B . Quantification of CRB3 staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 13 brains ) , LIS1 KO ( E12 . 5 , N = 4 brains ) ) and RAB6A / B dKO ( E15 . 5 , N = 4 brains ) . Data information : ( B , Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

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

B. Quantification of CRB3 staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=13 brains), LIS1 KO (E12.5, N=4 brains)) and RAB6A/B dKO (E15.5, N=4 brains). Data information: (B, Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.


C . N - Cadherin staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm .

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

C. N-Cadherin staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.


D . Quantification of N - Cadh staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 6 brains ) , LIS1 KO ( E12 . 5 , N = 3 brains ) and RAB6A / B dKO ( E15 . 5 , N = 3 brains ) . Data information : D ) Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

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

D. Quantification of N-Cadh staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=6 brains), LIS1 KO (E12.5, N=3 brains) and RAB6A/B dKO (E15.5, N=3 brains). Data information: D) Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.


A ) SH - SY5Y neuroblastoma cells were either cultivated in RPMI + 10 % FCS or starved in HBSS with or without addition of LY294002 or rapamycin . After 16 h PINK1 mRNA expression was determined by RT - qPCR and the PINK1 mRNA content of cells kept in RPMI + 10 % FCS was set as 1 . Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression , comparable to the positive control LY294002 ; n = 4 ; * expression changes compared to the PINK1 expression in RPMI + 10 % FCS : LY294002 : p < 0 . 01 ; rapamycin : p < 1 × 10 − 4 ; HBSS : p < 0 . 005 ; HBSS + LY294002 : p < 5 × 10 − 6 ; HBSS + rapamycin : p < 0 . 0005 ; # expression changes compared to the PINK1 expression in RPMI + 10 % FCS + LY294002 : p < 0 . 05 ; + expression changes compared to the PINK1 expression in RPMI + 10 % FCS + rapamycin : p < 0 . 0005 .

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

A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005.


B ) SH - SY5Y cells were either stably transduced with a control ( nt ) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5 % or 10 % FCS . Their PINK1 mRNA content was determined by RT - qPCR and PINK1 mRNA content of nt cells kept in medium with 10 % FCS was set as 1 . Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig . 1A , while stable PINK1 knockdown ( kd ) reduced PINK1 content under both conditions ; n = 3 ; * expression changes compared to the PINK1 expression in RPMI + 10 % FCS : PINK1 kd 10 % FCS p < 0 . 0005 ; PINK1 induction by 5 % FCS : p < 0 . 05 ; # expression changes compared to the PINK1 expression in RPMI + 5 % FCS : PINK1 kd 5 % FCS : p < 0 . 005 .

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

B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005.


C ) SH - SY5Y cells without ( nt ) or with stable PINK1 knockdown ( kd ) were kept in medium with 5 % FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1 . Afterwards the PINK1 63 kDa protein ( arrowhead ) and actin protein levels were determined by western blotting ( see representative gel on the right ) . The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells .

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

C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.


2A ) SH - SY5Y cells were starved for 2 h in HBSS with Bafilomycin ( + Baf ) or left untreated in RPMI + 5 % FCS ( - Baf ) . The LC3 - II and actin content were determined by western blotting . A representative blot is shown on the right , showing only LC3 - II bands in the Bafilomycin - treated samples . The LC3 - II bands of Bafilomycin treated samples were normalized to actin and the relative LC3 - II content of nt cells was set as 1 . Cells with stable PINK1 knockdown exhibited a reduced LC3 - II / actin ratio compared to the control ( nt ) cells ; n = 4 , p < 0 . 005 .

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

2A) SH-SY5Y cells were starved for 2 h in HBSS with Bafilomycin (+Baf) or left untreated in RPMI+5% FCS (- Baf). The LC3-II and actin content were determined by western blotting. A representative blot is shown on the right, showing only LC3-II bands in the Bafilomycin-treated samples. The LC3-II bands of Bafilomycin treated samples were normalized to actin and the relative LC3-II content of nt cells was set as 1. Cells with stable PINK1 knockdown exhibited a reduced LC3-II/actin ratio compared to the control (nt) cells; n = 4, p<0.005.


2B ) Cortical neurons from 3 different isolations ( 10 - 20 DIV ) of WT and PINK1 KO mice were either non - starved or starved for 2 h in HBSS and the LC3 - II and actin content was determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of WT and PINK1 KO mice , respectively , was set as 1 . Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3 - II / actin ratio compared to the WT cells ; n = 5 , p < 0 . 001 .

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

2B) Cortical neurons from 3 different isolations (10-20 DIV) of WT and PINK1 KO mice were either non-starved or starved for 2 h in HBSS and the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of WT and PINK1 KO mice, respectively, was set as 1. Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3-II/actin ratio compared to the WT cells; n = 5, p<0.001.


2C ) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA . After 48 h the LC3 - II and actin content was determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of cells transfected with scrambled siRNA was set as 1 . Transient PINK1 knockdown resulted in a reduced LC3 - II / actin ratio compared to the cells transfected with scrambled siRNA ; n = 6 ; p < 0 . 005 .

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

2C) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA. After 48 h the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with scrambled siRNA was set as 1. Transient PINK1 knockdown resulted in a reduced LC3-II/actin ratio compared to the cells transfected with scrambled siRNA; n = 6; p<0.005.


2D ) HeLa cells were transiently transfected with PINK1 - GFP , PINK1G309D - GFP or GFP . After 24 h cells were starved for 2 h in HBSS . Afterwards the LC3 - II and actin content were determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of cells transfected with GFP was set as 1 . Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3 - II / actin ratio compared to cells transfected with GFP ; n = 3 , PINK1 : p < 0 . 01 ; PINK1G309D : p < 0 . 05 .

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

2D) HeLa cells were transiently transfected with PINK1-GFP, PINK1G309D-GFP or GFP. After 24 h cells were starved for 2 h in HBSS. Afterwards the LC3-II and actin content were determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with GFP was set as 1. Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3-II/actin ratio compared to cells transfected with GFP; n = 3, PINK1: p<0.01; PINK1G309D: p<0.05.


3A ) Regular , non - transduced SH - SY5Y cells were cultivated either in RPMI + 10 % FCS or starved in HBSS medium for 24 and 40 h . The LAMP - 2 and actin content were determined by western blotting and the relative LAMP - 2 content of untreated cells was set as 1 . Starvation enhanced LAMP - 2protein levels ; n = 3 , p < 0 . 05 .

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

3A) Regular, non-transduced SH-SY5Y cells were cultivated either in RPMI+10% FCS or starved in HBSS medium for 24 and 40 h. The LAMP-2 and actin content were determined by western blotting and the relative LAMP-2 content of untreated cells was set as 1. Starvation enhanced LAMP-2protein levels; n = 3, p<0.05.


3B ) Transduced nt and PINK1 knockdown ( kd ) SH - SY5Y cells were cultivated either in RPMI + 5 % FCS or starved for 24 h and 40 h in HBSS . The LAMP - 2 and actin content were determined by western blotting . A representative blot after 40 h starvation in HBSS is shown on the right . The relative LAMP - 2 content of untreated cells was set as 1 . Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP - 2 / actin ratio compared to the nt cells ; n = 4 , p < 0 . 05 .

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

3B) Transduced nt and PINK1 knockdown (kd) SH-SY5Y cells were cultivated either in RPMI+5% FCS or starved for 24 h and 40 h in HBSS. The LAMP-2 and actin content were determined by western blotting. A representative blot after 40 h starvation in HBSS is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP-2/actin ratio compared to the nt cells; n = 4, p<0.05.


3C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp - 2 . Micrographs were taken with constant microscopical settings . LAMP - 2 positive signals in each cell were quantified with ImageJ as described in Material and Methods . Knockdown of PINK1 resulted in a decreased amount of LAMP - 2 positive stainings / cell after starvation ; n = 2 , at least 8 fields of view / condition , 89 - 124 cells / condition ; 24 h : p < 0 . 001 ; 40 h : p < 0 . 05 .

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

3C) nt and PINK1 knockdown (kd) SH-SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp-2. Micrographs were taken with constant microscopical settings. LAMP-2 positive signals in each cell were quantified with ImageJ as described in Material and Methods. Knockdown of PINK1 resulted in a decreased amount of LAMP-2 positive stainings/cell after starvation; n = 2, at least 8 fields of view/condition, 89-124 cells/condition; 24 h: p<0.001; 40 h: p<0.05.


3D ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were stained with the photo - reactive dye MTR and either irradiated for 45 min or left untreated . 24 h and 48 h after irradiation the LAMP - 2 and actin content were determined by western blotting . A representative blot 48 h after irradiation is shown on the right . The relative LAMP - 2 content of untreated cells was set as 1 . Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp - 2 / actin ratio compared to the control ( nt ) cells ; n = 5 , p < 0 . 001 .

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

3D) nt and PINK1 knockdown (kd) SH-SY5Y cells were stained with the photo-reactive dye MTR and either irradiated for 45 min or left untreated. 24 h and 48 h after irradiation the LAMP-2 and actin content were determined by western blotting. A representative blot 48 h after irradiation is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp-2/actin ratio compared to the control (nt) cells; n = 5, p<0.001.


3E ) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown ( kd ) SH - SY5Y cells cultivated in RPMI medium with 5 % FCS . The phosphatase activity was measured and normalized to the protein content . The phosphatase activity of nt cells was set as 1 . PINK1 knockdown mediated a reduction of lysosomal activity ; n = 3 ; p < 0 . 05 .

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

3E) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown (kd) SH-SY5Y cells cultivated in RPMI medium with 5% FCS. The phosphatase activity was measured and normalized to the protein content. The phosphatase activity of nt cells was set as 1. PINK1 knockdown mediated a reduction of lysosomal activity; n = 3; p<0.05.


4A ) Oxygen consumption rate ( OCR ) was measured in nt and PINK1 knockdown ( kd ) SH - SY5Y cells without ( nt ) or with PINK1 knockdown ( kd ) cultivated in RPMI medium with 5 % FCS . Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd , no significant changes were observed ; n = 1 ( quadruplicates ) .

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

4A) Oxygen consumption rate (OCR) was measured in nt and PINK1 knockdown (kd) SH-SY5Y cells without (nt) or with PINK1 knockdown (kd) cultivated in RPMI medium with 5% FCS. Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd, no significant changes were observed; n = 1 (quadruplicates).


4B ) Energy charge ( EC ) of nt and PINK1 knockdown ( kd ) SH - SY5Y cells grown in RPMI + 5 % FCS was determined as detailed in Material Methods . PINK1 knockdown resulted in a lower EC compared to nt cells ; n = 5 , p0 . 05 .

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

4B) Energy charge (EC) of nt and PINK1 knockdown (kd) SH-SY5Y cells grown in RPMI+5% FCS was determined as detailed in Material Methods. PINK1 knockdown resulted in a lower EC compared to nt cells; n = 5, p0.05.


4C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were grown for 2 months in RPMI + 5 % FCS and their population doublings were determined . Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth ; n = 1 ( triplicates ) ; * p < 0 . 05 ( day 12 ) - p < 1 × 10 − 6 ( day 61 ) .

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

4C) nt and PINK1 knockdown (kd) SH-SY5Y cells were grown for 2 months in RPMI+5% FCS and their population doublings were determined. Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth; n = 1 (triplicates); * p<0.05 (day 12) - p<1×10−6 (day 61).


4D ) SH - SY5Y cells were grown in RPMI + 5 % FCS and the amount of nt and PINK1 knockdown ( kd ) cells in S - phase was determined by BrdU incorporation . No differences in cell proliferation were visible between cells without ( nt ) and with PINK1 kd ; n = 3 .

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

4D) SH-SY5Y cells were grown in RPMI+5% FCS and the amount of nt and PINK1 knockdown (kd) cells in S-phase was determined by BrdU incorporation. No differences in cell proliferation were visible between cells without (nt) and with PINK1 kd; n = 3.


5A ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS or starved with HBSS for 12 h . DEVD cleavage as parameter for caspase - 3 activity was analyzed and normalized to the protein content . Cells with PINK1 knockdown demonstrated a significantly increased caspase - 3 activity under both conditions ; n = 4 ; RPMI + 5 % FCS : p < 0 . 05 ; HBSS : p < 0 . 01 .

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

5A) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS or starved with HBSS for 12 h. DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to the protein content. Cells with PINK1 knockdown demonstrated a significantly increased caspase-3 activity under both conditions; n = 4; RPMI+5% FCS: p<0.05; HBSS: p<0.01.


5B ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for the indicated time points . In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing .

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

5B) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing.


5C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for the indicated time points . For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH . Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation ; n = 4 ; 24 h : p < 0 . 005 .

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

5C) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH. Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation; n = 4; 24 h: p<0.005.


5D ) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP - Parkin or PINK1 - GFP or PINK1G309D - GFP . After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined . The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1 . Starvation - mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D - GFP but not by GFP or Parkin ; GFP : n = 5 , p < 0 . 005 ; Parkin : n = 3 , p < 0 . 05 ; PINK1 : n = 5 , ns ; PINK1G309D - GFP : n = 3 , ns .

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

5D) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP-Parkin or PINK1-GFP or PINK1G309D-GFP. After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined. The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1. Starvation-mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D-GFP but not by GFP or Parkin; GFP: n = 5, p<0.005; Parkin: n = 3, p<0.05; PINK1: n = 5, ns; PINK1G309D-GFP: n = 3, ns.


5E ) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA . After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase - 3 activity was analyzed and normalized to 1 , 000 , 000 cells . PINK1 knockdown increased caspase - 3 activity significantly , which was prevented by LC3 overexpression ; n = 3 ; p < 0 . 05 .

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

5E) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA. After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to 1,000,000 cells. PINK1 knockdown increased caspase-3 activity significantly, which was prevented by LC3 overexpression; n = 3; p<0.05.


5F ) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA . 48 h post transfection cells were either left untreated ( + medium ) or starved ( + HBSS ) for additional 24 h and afterwards the amount of adherent cells was determined . The amount of non - starved cells was set as 1 . PINK1 knockdown resulted in elevated cell loss , which was prevented by LC3 overexpression ; HeLa : n = 6 , p < 0 . 05 ; HeLa LC3 : n = 5 .

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

5F) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA. 48 h post transfection cells were either left untreated (+ medium) or starved (+HBSS) for additional 24 h and afterwards the amount of adherent cells was determined. The amount of non-starved cells was set as 1. PINK1 knockdown resulted in elevated cell loss, which was prevented by LC3 overexpression; HeLa: n = 6, p<0.05; HeLa LC3: n = 5.


A . Confocal images of mitochondrial morphology in wild - type ( WT ) and Drp1 - / - 293T cells transfected with empty vector ( left panel ) and Myc - hFis1 ( right panel ) , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

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

A. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Quantitative analyses of fragmented mitochondria size ( mean area ( μm2 ) per mitochondrion ) after Myc - hFis1 overexpression in WT and Drp1 - / - 293T cells using Image J software ( Particle analysis ) in three independent experiments . In each cell , only dispersed individual mitochondria were analyzed . The total number of mitochondria ( mito ) analyzed is indicated for each condition .

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

B. Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1-/- 293T cells using Image J software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition.


C . Quantitative analysis of mean mitochondria number per cell in WT and Drp1 - / - 293T cells overexpressing Myc - hFis1 using Image J software ( Particle analysis ) in three independent experiments .

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

C. Quantitative analysis of mean mitochondria number per cell in WT and Drp1-/- 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments.


E . Percentages of cells with indicated mitochondrial morphologies in WT and Drp1 - / - 293T cells transfected with empty vector ( control ) , Myc - hFis1 and either Myc - or GFP - tagged mutants as indicated in three independent experiments .

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

E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA ( control ) and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) as indicated . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

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

A. Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Western blot of Dyn2 , Myc - hFis1 , Drp1 and GAPDH in WT 293T cells collected from the experiments in ( A ) .

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

B. Western blot of Dyn2, Myc-hFis1, Drp1 and GAPDH in WT 293T cells collected from the experiments in (A).


C . Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector or Myc - hFis1 in three independent experiments .

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

C. Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector or Myc-hFis1 in three independent experiments.


D . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) . Representative examples of tubular , fragmented and tubular cluster phenotypes are indicated .

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

D. Confocal images of mitochondrial morphology in Drp1-/- 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented and tubular cluster phenotypes are indicated.


E . Western blot of Dyn2 , Myc - hFis1 and GAPDH in Drp1 - / - 293T cells collected from the experiments in ( D ) .

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

E. Western blot of Dyn2, Myc-hFis1 and GAPDH in Drp1-/- 293T cells collected from the experiments in (D).


F . Percentages of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells treated with scrambled siRNA or Dyn2 siRNA , followed by transfection with empty vector or Myc - hFis1 as indicated in three independent experiments .

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

F. Percentages of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells treated with scrambled siRNA or Dyn2 siRNA, followed by transfection with empty vector or Myc-hFis1 as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells transfected with scrambled siRNA ( control ) or with hFis1 - siRNA and then stained with MitoTracker ( red ) .

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

A. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red).


B . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells transfected with either Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

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

B. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with either Mfn1-Myc, Mfn2-Myc or OPA1-Myc, stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


C . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells transfected with either scrambled siRNA ( Ctr ) , hFis1 siRNA , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments for each condition ( n represents the number of cells analyzed ) .

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

C. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with either scrambled siRNA (Ctr), hFis1 siRNA, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments for each condition (n represents the number of cells analyzed).


D . hFis1 interacts with Mfn1 , Mfn2 and OPA1 as well as Drp1 , but not with Dyn2 at endogenous levels following chemical crosslinking . Wild - type ( WT ) and Drp1 - / - 293T cells were in vivo crosslinked with 1 % formaldehyde ( FA ) and cell lysates were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

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

D. hFis1 interacts with Mfn1, Mfn2 and OPA1 as well as Drp1, but not with Dyn2 at endogenous levels following chemical crosslinking. Wild-type (WT) and Drp1-/- 293T cells were in vivo crosslinked with 1% formaldehyde (FA) and cell lysates were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


hFis1 binds to Mfn1 , Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking . Cell lysates prepared from WT 293T ( E ) cells without chemical crosslinking were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

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

hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from WT 293T (E) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


F . hFis1 binds to Mfn1 , Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking . Cell lysates prepared from HeLa ( F ) cells without chemical crosslinking were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

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

F. hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from HeLa (F) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


G , H . Interaction of hFis1 with Mfn1 / 2 and with OPA1 are independent events . WT 293T cells were treated with control , OPA1 ( G ) or Mfn1 plus Mfn2 ( H ) siRNA , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

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

G, H. Interaction of hFis1 with Mfn1/2 and with OPA1 are independent events. WT 293T cells were treated with control, OPA1 (G) or Mfn1 plus Mfn2 (H) siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


I . Interactions between Mfn1 / 2 and OPA1 occur independent of hFis1 . WT 293T cells were treated with control or hFis1 siRNA , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to mouse normal IgG ( negative control ) or mouse anti - OPA1 antibody as indicated , followed by immunoblotting with indicated antibodies .

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

I. Interactions between Mfn1/2 and OPA1 occur independent of hFis1. WT 293T cells were treated with control or hFis1 siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG (negative control) or mouse anti-OPA1 antibody as indicated, followed by immunoblotting with indicated antibodies.


J . Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression . 293T cells were transfected with empty vector or Myc - hFis1 , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to mouse normal IgG or mouse anti - Mfn1 antibody , followed by immunoblotting with indicated antibodies .

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

J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies.


B . Interaction of different hFis1 mutants with Mfns and OPA1 . hFis1 - / - 293T cells were transfected with the full - length and the truncated hFis1 mutants as indicated , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with anti - Myc or anti - GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies .

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

B. Interaction of different hFis1 mutants with Mfns and OPA1. hFis1-/- 293T cells were transfected with the full-length and the truncated hFis1 mutants as indicated, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with anti-Myc or anti-GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies.


A , B . Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of exogenous hFis1 . WT 293T cells stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or with Myc - hFis1 as indicated and then treated with PEG for cell fusion . Mitochondrial fusion is indicated by co - localization of mitoGFP and mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1transfected . Insets represent magnification of the boxed areas in upper panel ( A ) . Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments for each condition and data summarized in ( B ) .

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

A, B. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or with Myc-hFis1 as indicated and then treated with PEG for cell fusion. Mitochondrial fusion is indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (B).


C , D . Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of exogenous hFis1 . WT 293T cells co - transfected with mito - PAGFP ( 0 . 5 µg ) , mito - DsRed ( 0 . 2 µg ) , and either empty vector ( 0 . 5 µg , the upper panel ) or hFis1 ( 0 . 5 µg , the lower panel ) were photoactivated in a small region of interest ( ROI ) ( white circle , 3 µm diameter ) in preactivation images of mitochondria seen by mito - DsRed ( red ) . After photoactivation , sequential Z - stack images with photoactivated GFP ( green ) and mitochondrial marker ( mito - DsRed ) were collected at indicated time points using series of Z - sections from the top to the cell bottom with intervals between sections set to 0 . 5 - 0 . 75 μm ( C ) . Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . The dilution rates ( percentage ) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation ( D ) .

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

C, D. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells co-transfected with mito-PAGFP (0.5 µg), mito-DsRed (0.2 µg), and either empty vector (0.5 µg, the upper panel) or hFis1 (0.5 µg, the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, sequential Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at indicated time points using series of Z-sections from the top to the cell bottom with intervals between sections set to 0.5-0.75 μm (C). Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of endogenous hFis1 . Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or hFis1 - siRNA as indicated and then co - cultured and fused using PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient in three independent experiments and data summarized in ( F ) .

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

E, F. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or hFis1-siRNA as indicated and then co-cultured and fused using PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient in three independent experiments and data summarized in (F).


G , H . Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated cells were co - transfected with mito - PAGFP and mito - DsRed and were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified ( H ) as described in ( D ) .

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

G, H. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified (H) as described in (D).


A , B . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of exogenous hFis1 . Drp1 - / - cells stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or Myc - hFis1 as indicated and then stimulated with PEG treatment for cell fusion , followed by immunostaining with anti - Myc antibody ( green ) . Mitochondrial fusion was indicated by co - localization of mitoGFP and mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1 transfected . Insets represent magnification of the boxed areas in upper panel ( A ) . Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments and summarized in ( B ) .

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

A, B. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or Myc-hFis1 as indicated and then stimulated with PEG treatment for cell fusion, followed by immunostaining with anti-Myc antibody (green). Mitochondrial fusion was indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1 transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (B).


C , D . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of exogenous hFis1 . Drp1 - / - cells co - transfected with mito - PAGFP , mito - DsRed , and either empty vector ( the upper panel ) or hFis1 ( the lower panel ) were photoactivated in a small region of interest ( ROI ) ( white circle , 3 µm diameter ) as indicated in preactivation images of mitochondria seen by mito - DsRed ( red ) . After photoactivation , Z - stack images with photoactivated GFP ( green ) and mitochondrial marker ( mito - DsRed ) were collected at times 40 sec , 15 , 30 , 45 min as indicated ( C ) . Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . The dilution rates ( percentage ) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation ( D ) .

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

C, D. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells co-transfected with mito-PAGFP, mito-DsRed, and either empty vector (the upper panel) or hFis1 (the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) as indicated in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at times 40 sec, 15, 30, 45 min as indicated (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of endogenous hFis1 . Drp1 - / - cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or with hFis1 - siRNA as indicated and then co - cultured and fused by PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments and summarized in ( F ) .

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

E, F. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or with hFis1-siRNA as indicated and then co-cultured and fused by PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (F).


G , H . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T cells monitored by the mito - PAGFP - based fusion assay in the presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated cells were co - transfected with mito - PAGFP and mito - DsRed and were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito - PAGFP ( H ) as described in ( D ) .

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

G, H. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells monitored by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP (H) as described in (D).


A , B . GTP hydrolysis activities of Mfn1 , Mfn2 , Mfn2K109A and OPA1 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined by measuring the concentration of free phosphate ( Pi ) released from GTP . Myc - tagged Mfn1 , Mfn2 , Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti - Myc agarose beads . GTPase activities of immunopurified Myc - tagged Mfn1 , Mfn2 , Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST - hFis1 or GST protein ( A ) . The input levels of immunopurified Mfn1 - Myc , Mfn2 - Myc and OPA1 - Myc ( left panel ) as well as recombinant GST - hFis1 ( ~ 42 kDa ) and GST ( ~ 26 kDa ) ( right panel ) were assessed by immunoblotting with indicated antibodies ( B ) .

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

A, B. GTP hydrolysis activities of Mfn1, Mfn2, Mfn2K109A and OPA1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined by measuring the concentration of free phosphate (Pi) released from GTP. Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti-Myc agarose beads. GTPase activities of immunopurified Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST-hFis1 or GST protein (A). The input levels of immunopurified Mfn1-Myc, Mfn2-Myc and OPA1-Myc (left panel) as well as recombinant GST-hFis1 (~42 kDa) and GST (~26 kDa) (right panel) were assessed by immunoblotting with indicated antibodies (B).


C , D . GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1 - / - 293T cells and immunopurified by protein G beads pre - incubated with Drp1 antibody . GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein ( C ) . The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti - Drp1 antibody ( D ) .

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

C, D. GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1-/- 293T cells and immunopurified by protein G beads pre-incubated with Drp1 antibody. GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein (C). The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti-Drp1 antibody (D).


E , F . GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Endogenous Dyn2 in Drp1 - / - 293T cells was immunopurified by protein G beads pre - incubated with anti - Dyn2 antibody . GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST - hFis1 or GST protein ( E ) . The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti - Dyn2 antibody ( F ) .

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

E, F. GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Endogenous Dyn2 in Drp1-/- 293T cells was immunopurified by protein G beads pre-incubated with anti-Dyn2 antibody. GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST-hFis1 or GST protein (E). The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti-Dyn2 antibody (F).


G . Summary of the effects of pro - fusion GTPases ( Mfns and OPA1 ) on hFis1 - induced mitochondrial fragmentation in Drp1 - / - cells . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells co - expressing Myc - hFis1 and either empty vector , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments . n represents the number of cells analyzed . Corresponding confocal images shown in Fig EV4 .

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

G. Summary of the effects of pro-fusion GTPases (Mfns and OPA1) on hFis1-induced mitochondrial fragmentation in Drp1-/- cells. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells co-expressing Myc-hFis1 and either empty vector, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments. n represents the number of cells analyzed. Corresponding confocal images shown in Fig EV4.


A . Ablation of OPA1 using CRISPR - Cas9 gene editing in Drp1 - / - 293T cells was confirmed by Western blotting analysis .

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

A. Ablation of OPA1 using CRISPR-Cas9 gene editing in Drp1-/- 293T cells was confirmed by Western blotting analysis.


B . Confocal images of mitochondrial morphology in Drp1 - / - ( left panel ) and Drp1 / OPA1DKO 293T ( right panel ) cells . ( Aggr / frag : aggregation / fragmentation of mitochondria ; Bulb / tubular : bulb - like / tubular mitochondria ) .

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

B. Confocal images of mitochondrial morphology in Drp1-/- (left panel) and Drp1/OPA1DKO 293T (right panel) cells. (Aggr/frag: aggregation/fragmentation of mitochondria; Bulb/tubular: bulb-like/tubular mitochondria).


C . Confocal images of mitochondrial morphology in Drp1 / OPA1DKO 293T cells treated with the indicated siRNAs , combinations of siRNAs or with Myc - hFis1 plasmid ( lower three panels ) compared to mitochondrial morphology in Drp1 - / - 293T cells overexpressing Myc - hFis1 or not ( upper panel ) .

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

C. Confocal images of mitochondrial morphology in Drp1/OPA1DKO 293T cells treated with the indicated siRNAs, combinations of siRNAs or with Myc-hFis1 plasmid (lower three panels) compared to mitochondrial morphology in Drp1-/- 293T cells overexpressing Myc-hFis1 or not (upper panel).


D . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells transfected with empty vector or Myc - hFis1 plasmid , and in Drp1 / OPA1DKO 293T cells treated with the indicated siRNA , combinations of siRNA or Myc - hFis1 plasmid . The data were collected from three independent experiments . Total cell numbers ( n ) used for statistical analysis are indicated for each condition .

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

D. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with empty vector or Myc-hFis1 plasmid, and in Drp1/OPA1DKO 293T cells treated with the indicated siRNA, combinations of siRNA or Myc-hFis1 plasmid. The data were collected from three independent experiments. Total cell numbers (n) used for statistical analysis are indicated for each condition.


E . Knockdown of the indicated proteins by siRNA in Drp1 / OPA1DKO 293T cells was confirmed by Western blotting analysis .

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

E. Knockdown of the indicated proteins by siRNA in Drp1/OPA1DKO 293T cells was confirmed by Western blotting analysis.