F - I Confocal of transfected with mito - YFP treated with DMSO ( F , control ) or IU1 ( G ) for 24h . Fragmentation index ( H ) total mitochondrial volume / were measured represented in ( I ) , mean ± . At least 35 were analysed from 3 different experimental conditions . Scale bar 10 μm

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F-I Confocal images of cells transfected with mito-YFP and treated with DMSO (F, control) or IU1 (G) for 24h. Fragmentation index (H) and total mitochondrial volume / cell were measured and represented in (I), mean ±SEM. At least 35 cells were analysed from 3 different experimental conditions. Scale bar 10 μm


J - L Mito - YFP transfected were treated with IU1 ( 100 μM , 24h ) , fixed immuno labelled for LC3 . Number of LC3 dots / ( K ) LC3 dot overlapping mitochondria ( L ) were counted . At least 35 from 3 independent experiments were analysed . Scale bar : 10 μm

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J-L Mito-YFP transfected cells were treated with IU1 (100 μM, 24h), fixed and immuno labelled for LC3. Number of LC3 dots / cell (K) and LC3 dot overlapping mitochondria (L) were counted. At least 35 cells from 3 independent experiments were analysed. Scale bar: 10 μm


M - N were transfected with mito - keima treated with DMSO or IU1 for 24h subjected to FACS , counting 10 , 000 per experiment . ( M ) represents shift of signal in IU1 treated when excited at 560 nm ( false colour ) . ( N ) represents average of mito - keima when excited at 560 nm between 560 : 405 nm . Graphs represent mean ± from 3 different sets of

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M-N Cells were transfected with mito-keima and treated with DMSO or IU1 for 24h and subjected to FACS analysis, counting 10,000 cells per experiment. (M) represents the shift of the signal intensity in IU1 treated cells when excited at 560 nm (false colour). (N) represents the average intensity of the mito-keima protein when excited at 560 nm and the ratio between 560: 405 nm. Graphs represent mean ± SEM from 3 different sets of experiments


A - B Western blot of indicated in lysates from SH - SY5Y , treated with different concentrations of IU1 ( 1 - 100 μM ) for 24h . Bar graphs represents mean ± . Blots are representative of three independent experiments . ANOVA followed by Dunnets

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A-B Western blot analysis of the indicated proteins in cell lysates from SH-SY5Y cells, treated with different concentrations of IU1 (1-100 μM) for 24h. Bar graphs represents mean ± SEM. Blots are representative of three independent experiments. ANOVA followed by Dunnets test


C - D Representative of MEF knocked out for indicated mitochondrial dynamics transfected with mito - YFP ( C ) with / without IU1 treatment ( 100 μM , 48h ) . Scale bar 10 μm . ( D ) Mitochondrial volume / was measured as described represented as mean ± . At least 35 were

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C-D Representative images of MEF cells knocked out for the indicated mitochondrial dynamics protein and transfected with mito-YFP (C) with / without IU1 treatment (100 μM, 48h). Scale bar 10 μm. (D) Mitochondrial volume / cell was measured as described and represented as mean ±SEM. At least 35 cells were evaluated


E - F Western blot of HSP60 ATP5a , as mitochondrial marker in indicated knock out MEF ( with / without indicated DRP1 variant ) after 48h of IU1 treatment . Bar graphs represent mean ± , n = at least 3 . Student ' s t ;

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E-F Western blot analysis of HSP60 and ATP5a, as mitochondrial marker in the indicated knock out MEF cells (with / without the indicated DRP1 variant) after 48h of IU1 treatment. Bar graphs represent mean ± SEM, n= at least 3. Student's t test;


A - D Wild type MEF ( A ) , Hela ( B ) PINK1 KO MEF ( C ) were treated with IU1 ( 48h , 100 μM ) electron microscope were evaluated for autophagic vesicle formation . ( D ) bar graphs represents mean ± number of autophagosomes autolysosome / from 3 different set of experiments ; at least 40 were analysed from each

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A-D Wild type MEF (A), Hela (B) and PINK1 KO MEF (C) cells were treated with IU1 (48h, 100 μM) and electron microscope images were evaluated for autophagic vesicle formation. (D) bar graphs represents mean± SEM number of autophagosomes and autolysosome / cell from 3 different set of experiments; at least 40 cells were analysed from each group


E Western blot of HSP60 with or without IU1 treatment in indicated type . Bar graphs represent mean ± . N = at least 3 independent experiments . Student ' s t . * P ≤ 0 . 05 , * * P ≤ 0 . 01

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E Western blot analysis of HSP60 protein with or without IU1 treatment in the indicated cell type. Bar graphs represent mean ± SEM. N= at least 3 independent experiments. Student's t test. *P ≤ 0.05, **P ≤ 0.01


A - B SH - SY5Y were immunostained for ATP5a ( green ) 20S proteasome ( red ) after IU1 treatment ( 6h - 24h ) . Scale bar 10 μm . Colour mapping of co - localisation of HSP60 20S proteasome from selected regions were generated by image j represented in bottom panel . of co - localisation is represented in colour map scale at bottom of panel . ( B ) 20S proteasome subunit mitochondria co - localisation was quantified by measuring Mander ' s coefficient . Bar graphs represent mean fold change ( mean ± ) from 3 different experiments . At least 30 were evaluated for calculations . ANOVA followed by Dunnet ' s

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A-B SH-SY5Y cells were immunostained for ATP5a (green) and 20S proteasome core (red) after IU1 treatment (6h-24h). Scale bar 10 μm. Colour mapping of the co-localisation of HSP60 and 20S proteasome from the selected regions were generated by image j and represented in the bottom panel. Intensity of the co-localisation is represented in the colour map scale at the bottom of the panel. (B) 20S proteasome subunit and mitochondria co-localisation was quantified by measuring Mander's coefficient. Bar graphs represent mean fold change (mean ± SEM) from 3 different experiments. At least 30 cells were evaluated for the calculations. ANOVA followed by Dunnet's test


C Electron microscopy showing subcellular localization of immuno - gold labelled 20S subunit of proteasome after 12h or 24h of IU1 treatment . Experiments were repeated twice in two biological replicates with similar results . contrast in magnified region is enhanced from original image to highlight immunogold labelled signals

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C Electron microscopy images showing subcellular localization of immuno- gold labelled 20S subunit of proteasome complex after 12h or 24h of IU1 treatment. Experiments were repeated twice in two biological replicates with similar results. The contrast in the magnified region is enhanced from the original image to highlight the immunogold labelled signals


D - E proximity ligation for Prohibitin2 ( PHB2 ) LC3 after 24h IU1 treatment . showing signal are merged with bright field to signal dots ( cyan false colour ) as depicted in figure . In inset only signal is shown , without merging with bright field from selected regions . Scale bar 10 μm . ( E ) Bar graphs represent mean ± of number of signal dots per from 3 different experiments . Student ' s t ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001

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D-E Protein proximity ligation assay for Prohibitin2 (PHB2) and LC3 after 24h IU1 treatment. Images showing signal are merged with bright field images to count the signal dots (cyan false colour) as depicted in the figure. In the inset only fluorescence signal is shown, without merging with the bright field from the selected regions. Scale bar 10 μm. (E) Bar graphs represent mean ±SEM of the number of signal dots per cell from 3 different experiments. Student's t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001


Electron microscope of mitochondria from SH - SY5Y e , as indicated in figure , with / without USP14 inhibition / knock down . Arrow heads in IU1 treated groups indicate mitochondrial rupture point . Bar graphs represent mean ( ± ) of ruptured mitochondria from at least 3 different experiments . For , total 150 - 300 mitochondria for control group 150 - 190 mitochondria from IU1 treated groups were

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Electron microscope images of mitochondria from SH-SY5Y e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Representative immunoblot of isolated mitochondria from indicated groups treated with / without trypsin probed for HSP60 , ATP5a TOM20 . Bar graphs represent mean ± of of densitometric of indicated ( with : without trypsin ) from at least 3 independent

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Representative immunoblot of isolated mitochondria from the indicated groups treated with / without trypsin and probed for HSP60, ATP5a and TOM20. Bar graphs represent mean ± SEM of the ratio of densitometric levels of the indicated proteins (with : without trypsin) from at least 3 independent experiments


Electron microscope of mitochondria fro MEF WT , MEF PINK1 K as indicated in figure , with / without USP14 inhibition / knock down . Arrow heads in IU1 treated groups indicate mitochondrial rupture point . Bar graphs represent mean ( ± ) of ruptured mitochondria from at least 3 different experiments . For , total 150 - 300 mitochondria for control group 150 - 190 mitochondria from IU1 treated groups were

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Electron microscope images of mitochondria fro MEF WT, MEF PINK1 K as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Electron microscope of mitochondria fro Hela an e , as indicated in figure , with / without USP14 inhibition / knock down . Arrow heads in IU1 treated groups indicate mitochondrial rupture point . Bar graphs represent mean ( ± ) of ruptured mitochondria from at least 3 different experiments . For , total 150 - 300 mitochondria for control group 150 - 190 mitochondria from IU1 treated groups were

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Electron microscope images of mitochondria fro Hela an e, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments. For cells, total 150-300 mitochondria for the control group and 150-190 mitochondria from the IU1 treated groups were counted


Electron microscope of mitochondria fro Drosophila thoracic muscle , as indicated in figure , with / without USP14 inhibition / knock down . Arrow heads in IU1 treated groups indicate mitochondrial rupture point . Bar graphs represent mean ( ± ) of ruptured mitochondria from at least 3 different experiments For flies , more than 200 mitochondria from each group were counted from 5 different

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Electron microscope images of mitochondria fro Drosophila thoracic muscle, as indicated in the figure, with / without USP14 inhibition/knock down. Arrow heads in the IU1 treated groups indicate the mitochondrial rupture point. Bar graphs represent mean (± SEM) of percentage ruptured mitochondria from at least 3 different experiments For flies, more than 200 mitochondria from each group were counted from 5 different experiments


A Life span of flies of indicated genetic background . At least 50 flies were monitored from each group . Log - rank ( Mantel - Cox , Mantel - Haenszel , p < 0 . 0001 )

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A Life span analysis of flies of the indicated genetic background. At least 50 flies were monitored from each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)


B Graph bar shows mean ± of climbing performance of flies of indicated genotypes . n = 4 - 7 . ANOVA followed by Newman Keuls

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B Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. n=4-7. ANOVA followed by Newman Keuls test


C Quantitative of respiratory fitness of isolated mitochondria extracted from flies of indicated genotypes . Graph shows respiratory control rate ( RCR ) calculated as described in materials methods . Graphs represent mean ± . N = 4 - 5 , ANOVA followed by Newman Keuls

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C Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows respiratory control rate (RCR) calculated as described in materials and methods. Graphs represent mean± SEM. N=4-5, ANOVA followed by Newman Keuls test


D Dopamine content in fly heads ( 15 day old ) was measured by HPLC fold change values are represented in scatter graph . Graphs represent mean ± . N = 14 , 11 11 fly heads for control , PINK1 PINK1 + USP14 KD fly respectively . ANOVA followed by Newman Keuls

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D Dopamine content in the fly heads (15 day old) was measured by HPLC and the individual fold change values are represented in the scatter graph. Graphs represent mean± SEM. N= 14, 11 and 11 fly heads for control, PINK1 and PINK1+USP14 KD fly respectively. ANOVA followed by Newman Keuls test


E Enlarged representative of flight muscle mitochondria of indicated genotypes . experiment was repeated 3 times in replicates . Bar graphs represent of electron dense mitochondria ( represented as grey bar ) depolarised mitochondrial with ruptured cristae ( represented as bars ) from indicated genotypes

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E Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated 3 times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and depolarised mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


( A ) Life span of flies of indicated genetic background . At least 50 flies were analysed for each group . Log - rank ( Mantel - Cox , Mantel - Haenszel , p < 0 . 0001 )

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(A) Life span analysis of flies of the indicated genetic background. At least 50 flies were analysed for each group. Log-rank test (Mantel-Cox test, Mantel-Haenszel test, p < 0.0001)


( B ) Graph bar shows mean ± of climbing performance of flies of indicated genotypes . One way ANOVA , Newman Keuls , n = 3

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(B) Graph bar shows mean± SEM of the climbing performance of flies of the indicated genotypes. One way ANOVA, Newman Keuls test, n=3


( C ) Quantitative of respiratory fitness of isolated mitochondria extracted from flies of indicated genotypes . Graph shows ( mean ± , n = 3 independent experiments ) respiratory control rate ( RCR ) calculated as described . One way ANOVA , Newman Keuls

(C) Quantitative analysis of respiratory fitness of isolated mitochondria extracted from flies of the indicated genotypes. Graph shows (mean± SEM, n=3 independent experiments) respiratory control rate (RCR) calculated as described. One way ANOVA, Newman Keuls test


( D ) Enlarged representative of flight muscle mitochondria of indicated genotypes . experiment was repeated three times in replicates . Bar graphs represent of electron dense mitochondria ( represented as grey bar ) mitochondrial with ruptured cristae ( represented as bars ) from indicated genotypes

(D) Enlarged TEM representative images of flight muscle mitochondria of the indicated genotypes. The experiment was repeated three times in replicates. Bar graphs represent quantification of electron dense mitochondria (represented as grey bar) and mitochondrial with ruptured cristae (represented as white bars) from the indicated genotypes


( a ) Identification of Beclin1 as a substrate of ROCK1 via affinity purification . Left panel : HeLa growing in control ( C : nutrient - rich media ) or starved in HBSS ( H ) for 4 h were lysed subjected to IP with ROCK1 cross - linked beads . bound were eluted detected by SDS - PAGE , followed by silver that presents precipitated identified by LC - MS / MS . Right panel : HeLa grown in control media or HBSS ( 4 h ) were collected , whole lysates prepared immunoprecipitated with ROCK1 cross - linked beads . resulting immune complexes were divided into two , were subjected to SDS - PAGE analysed for Beclin1 ( MES buffer ) ROCK1 ( MOPS buffer ) . Inputs IP were analysed by western blotting . HBSS - treated extracts were used for IgG IP controls . Whole lysates ( 10 % ) were run for inputs . Total lysates were rerun analysed for LC3 β - actin .

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(a) Identification of Beclin1 as a substrate of ROCK1 via affinity purification. Left panel: HeLa cells growing in control (C: nutrient-rich media) or starved in HBSS (H) for 4 h were lysed and subjected to IP with ROCK1 cross-linked beads. Proteins bound were eluted and detected by SDS-PAGE, followed by silver staining that presents precipitated proteins identified by LC-MS/MS. Right panel: HeLa cells grown in control media or HBSS (4 h) were collected, whole cell lysates prepared and immunoprecipitated with ROCK1 cross-linked beads. The resulting immune complexes were divided into two, and were subjected to SDS-PAGE and analysed for Beclin1 (MES buffer) and ROCK1 (MOPS buffer). Inputs and IP were analysed by western blotting. HBSS-treated cell extracts were used for IgG IP controls. Whole cell lysates (10%) were run for inputs. Total cell lysates were rerun and analysed for LC3 and β-actin.


( b ) Co - IP was performed with 293T whole extracts by using ROCK1 - crosslinked agarose , resulting IP was blotted with indicated antibodies . 293T transfected with Flag - Beclin1 for 36 h , cultured in control media or starved ( 4 h ) , were immunoprecipitated with anti - Flag beads . Inputs immune complexes were resolved detected . Total lysates were rerun examined for LC3 β - actin expression .

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(b) Co-IP was performed with 293T whole cell extracts by using ROCK1-crosslinked agarose, and the resulting IP was blotted with indicated antibodies. 293T cells transfected with Flag-Beclin1 for 36 h, cultured in control media or starved (4 h), were immunoprecipitated with anti-Flag beads. Inputs and immune complexes were resolved and detected. Total cell lysates were rerun and examined for LC3 and β-actin expression.


( c ) Interaction of YFP - ROCK1 CFP - Beclin1 was examined by using live spinning disk microscopy FRET in live . Graph represents FRETAverage changes ( YFP - ROCK1 / CFP - Beclin1 ) upon starvation . Data represents mean ± s . e . m . ; n = 6 . Three different experiments were performed . For control , right top graph represents CFP over time ( corrected for photobleach ) , right bottom right represents YFP ( corrected for photobleach ) . In both control FRET experiments , no significant change was observed in , in presence or absence of HBSS medium . P values were calculated using Student ' s t - .

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(c) Interaction of YFP-ROCK1 and CFP-Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells. Graph represents FRETAverage ratio changes (YFP-ROCK1/CFP-Beclin1) upon starvation. Data represents mean±s.e.m.; n=6. Three different experiments were performed. For the control, the right top graph represents CFP intensity over time (corrected for photobleach), and the right bottom right represents YFP intensity (corrected for photobleach). In both control FRET experiments, no significant change was observed in fluorescence, in the presence or absence of HBSS medium. P values were calculated using Student's t-test.


( a ) Inhibition of ROCK1 activity ablates interaction between Beclin1 ROCK1 . HeLa were untreated ( H2O ) or treated with 10 μM ROCK1 inhibitor , Y27632 , for 8 h then cultured in control media ( + ) or HBSS ( − ) for 4 h . were collected , lysed immunoprecipitated with Beclin1 ( left panel ) or ROCK1 ( right panel ) antibody . Left panel : immune complexes were divided into two ( ROCK1 - MOPS buffer Beclin1 - MES buffer ) probed for ROCK1 Beclin1 . Relative values of ROCK1 versus Beclin1 are shown .

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(a) Inhibition of ROCK1 activity ablates the interaction between Beclin1 and ROCK1. HeLa cells were untreated (H2O) or treated with 10 μM ROCK1 inhibitor, Y27632, for 8 h and then cultured in control media (+) or HBSS (−) for 4 h. Cells were collected, lysed and immunoprecipitated with Beclin1 (left panel) or ROCK1 (right panel) antibody. Left panel: immune complexes were divided into two (ROCK1-MOPS buffer and Beclin1-MES buffer) and probed for ROCK1 and Beclin1. Relative values of ROCK1 versus Beclin1 are shown.


( b ) An increase in ROCK1 - mediated phosphorylation of MYPT1 upon starvation . HeLa EJ were starved in HBSS for indicated time points endogenous ROCK1 was immunoprecipitated . An in vitro kinase was performed using MYPT1 as substrate immune - complexed ROCK1 . were resolved visualized with Coomassie ( top ) , western blot ( middle ) autoradiography ( bottom ) . Inputs were examined for ROCK1 β - actin .

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(b) An increase in ROCK1-mediated phosphorylation of MYPT1 upon starvation. HeLa and EJ cells were starved in HBSS for indicated time points and endogenous ROCK1 was immunoprecipitated. An in vitro kinase assay was performed using MYPT1 as substrate and immune-complexed ROCK1. Proteins were resolved and visualized with Coomassie staining (top), western blot analysis (middle) and autoradiography (bottom). Inputs were examined for ROCK1 and β-actin.


( c ) HeLa incubated in HBSS were collected endogenous phosphorylation of MYPT1 was determined by western blotting . same blot was then washed in TBST for 30 min , blocked in 5 % milk was analysed for total MYPT1 as a control . Extracts from this experiment were rerun on 7 . 5 % gel analysed for ROCK1 β - actin .

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(c) HeLa cells incubated in HBSS were collected and endogenous phosphorylation of MYPT1 was determined by western blotting. The same blot was then washed in TBST for 30 min, blocked in 5% milk and was analysed for total MYPT1 as a control. Extracts from this experiment were rerun on 7.5% gel and analysed for ROCK1 and β-actin.


( d ) ROCK activity is increased upon starvation . lines ( HeLa EJ ) grown in control ( + Glu ) or starved with HBSS for 1 3 h were lysed , extracts normalized ROCK kinase activity was measured by performing enzyme - linked immunosorbent . Graphs represent ROCK activity in starved plotted as of ROCK1 activity in control ( glucose - rich ) . Data were obtained from triplicates performed at same time ( mean ± s . d . ) . P - value was calculated using Student ' s t - .

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(d) ROCK activity is increased upon starvation. Cell lines (HeLa and EJ) grown in control (+Glu) or starved with HBSS for 1 and 3 h were lysed, extracts normalized and ROCK kinase activity was measured by performing enzyme-linked immunosorbent assay. Graphs represent ROCK activity in starved cells plotted as percent of ROCK1 activity in control (glucose-rich) cells. Data were obtained from triplicates performed at the same time (mean±s.d.). P-value was calculated using Student's t-test.


( e ) ROCK1 activity is not dependent on Beclin1 . siControl ( siCont . ) siBeclin1 knockdown were incubated in starvation media ( HBSS ) for indicated time . Resulting extracts were used to confirm Beclin1 knockdown ROCK1 expression ( left panel ) . extracts from same experiment were used for enzyme - linked immunosorbent measuring ROCK activity ( right panel ) . Graph represents mean ± s . d . of duplicate samples read at same time ( Student ' s t - ; NS , not significant ) .

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(e) ROCK1 activity is not dependent on Beclin1. siControl (siCont.) and siBeclin1 knockdown cells were incubated in starvation media (HBSS) for indicated time. Resulting cell extracts were used to confirm Beclin1 knockdown and ROCK1 expression (left panel). Cell extracts from the same experiment were used for enzyme-linked immunosorbent assay measuring ROCK activity (right panel). Graph represents mean±s.d. of duplicate samples read at the same time (Student's t-test; NS, not significant).


( a ) effect of ROCK1 depletion on autophagy marker , LC3II . MEF ROCK1flox / flox were generated treated with Con or Ad - Cre for 72 h . were then incubated in nutrient - rich ( + Glucose ) or HBSS media for 2 h . extracts were resolved by SDS - PAGE ROCK1 KO was confirmed by western blotting ( left panel ) . same blot was used for LC3 β - actin expression . from same experiment were fixed with chilled acetone immunofluroscence was performed against endogenous LC3 ( red ) . Blue represents DAPI . Scale bar , 20 μm .

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(a) The effect of ROCK1 depletion on autophagy marker, LC3II. MEF ROCK1flox/flox cells were generated and treated with Con or Ad-Cre for 72 h. Cells were then incubated in nutrient-rich (+Glucose) or HBSS media for 2 h. Cell extracts were resolved by SDS-PAGE and ROCK1 KO was confirmed by western blotting (left panel). The same blot was used for LC3 and β-actin expression. Cells from the same experiment were fixed with chilled acetone and immunofluroscence was performed against endogenous LC3 (red). Blue represents nuclear DAPI staining. Scale bar, 20 μm.


( b ) EJ ( left ) with a stable knockdown using shCont . or shROCK1 # 1 were incubated in control ( + glucose ) or nutrient - free ( HBSS ) medium ( − glucose ) for 24 h lysed . Lysates were subjected to western blotting with antibodies indicated . HeLa ( right ) with a transient knockdown using siCont . siROCK1 # 2 for 30 h were further incubated in control or HBSS media for 8 h . Whole extracts were prepared , resolved by SDS - PAGE analysed for ROCK1 , LC3 β - actin . Relative ratios of LC3II / LC3I are shown .

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(b) EJ cells (left) with a stable knockdown using shCont. or shROCK1#1 were incubated in control (+glucose) or nutrient-free (HBSS) medium (−glucose) for 24 h and lysed. Lysates were subjected to western blotting with antibodies indicated. HeLa cells (right) with a transient knockdown using siCont. and siROCK1#2 for 30 h were further incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and analysed for ROCK1, LC3 and β-actin. Relative ratios of LC3II/LC3I are shown.


( c ) HeLa were treated with H2O or 10 μM ROCK1 inhibitor , Y27632 , for 8 h . were then cultured in high glucose ( 0 h ) or starved for in HBSS nutrient - free media for 6 h . One hour before collection , were incubated in dimethylsulphoxide ( control ) or 0 . 1 μM bafilomycin - A1 . Whole lysates were resolved by SDS - PAGE were analysed by western blotting with indicated antibodies .

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(c) HeLa cells were treated with H2O or 10 μM ROCK1 inhibitor, Y27632, for 8 h. Cells were then cultured in high glucose (0 h) or starved for in HBSS nutrient-free media for 6 h. One hour before collection, cells were incubated in dimethylsulphoxide (control) or 0.1 μM bafilomycin-A1. Whole cell lysates were resolved by SDS-PAGE and were analysed by western blotting with indicated antibodies.


( d ) HeLa incubated with H2O or 10 μM Y27632 for 8 h were washed with PBS cultured in control ( + Glu ) or HBSS media ( − Glu ) for an additional 8 h . were fixed in acetone immunofluorescence was performed against endogenous LC3 antibody ( green ) . Scale bar , 20 μm . Graph represents % of LC3 / ( mean ± s . d . ) , n = 25 ; ( Student ' s t - ) * * P = 0 . 00041 .

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(d) HeLa cells incubated with H2O or 10 μM Y27632 for 8 h were washed with PBS and cultured in control (+Glu) or HBSS media (−Glu) for an additional 8 h. Cells were fixed in acetone and immunofluorescence was performed against endogenous LC3 antibody (green). Scale bar, 20 μm. Graph represents % of LC3 punctae/cell (mean±s.d.), n=25 cells; (Student's t-test) **P=0.00041.


( e ) Transmission electron of EJ with stable shCont . or shROCK1 # 1 knockdown , cultured in control or HBSS medium for 16 h . Arrow bars indicate autophagic vacuoles . Scale bar , 500 nm .

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(e) Transmission electron micrographs of EJ cells with stable shCont. or shROCK1#1 knockdown, cultured in control or HBSS medium for 16 h. Arrow bars indicate autophagic vacuoles. Scale bar, 500 nm.


( a ) Inhibition of ROCK1 kinase activity prevents metabolic stress - mediated phosphorylation of Beclin1 . HeLa left untreated or treated with a ROCK1 inhibitor , Y27632 ( 10 μM ) , were starved of glucose ( 4 h ) as indicated , endogenous Beclin1 was immunoprecipitated blotted against phospho - Ser / Thr antibody . Whole lysates were run as inputs for Beclin1 β - actin .

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(a) Inhibition of ROCK1 kinase activity prevents metabolic stress-mediated phosphorylation of Beclin1. HeLa cells left untreated or treated with a ROCK1 inhibitor, Y27632 (10 μM), were starved of glucose (4 h) as indicated, and endogenous Beclin1 was immunoprecipitated and blotted against phospho-Ser/Thr antibody. Whole cell lysates were run as inputs for Beclin1 and β-actin.


( b ) Phosphorylation of Beclin1 by ROCK1 . Recombinant ROCK1 was used for in vitro kinase in presence or absence of Y27632 , using recombinant His - Beclin1 ( left ) or Flag - Beclin1 immunopurified from transfected HeLa ( right ) . were resolved by SDS - PAGE ; phosphorylated was visualized with autoradiography , Beclin1 by Coomassie .

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(b) Phosphorylation of Beclin1 by ROCK1. Recombinant ROCK1 was used for in vitro kinase assay in the presence or absence of Y27632, using recombinant His-Beclin1 (left) or Flag-Beclin1 immunopurified from transfected HeLa cells (right). Proteins were resolved by SDS-PAGE; phosphorylated protein was visualized with autoradiography, and Beclin1 by Coomassie staining.


( c ) Identification of ROCK1 - mediated phosphorylation domains in Beclin1 . indicated Flag - Beclin1 fragments were purified from HeLa used as substrates for in vitro ROCK1 kinase . 32P - autoradiogram ( centre ) analysed phosphorylation western blotting ( WB ; right ) determined . Schematic representation of Beclin1 domain structure deletion constructs are shown ( left ) .

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(c) Identification of ROCK1-mediated phosphorylation domains in Beclin1. The indicated Flag-Beclin1 fragments were purified from HeLa cells and used as substrates for in vitro ROCK1 kinase assay. 32P-autoradiogram (centre) analysed phosphorylation and western blotting (WB; right) determined protein levels. Schematic representation of Beclin1 domain structure and deletion constructs are shown (left).


( d ) Identification of phosphorylation site on Beclin1 . Representation of point mutations in different domains of Beclin1 are shown ( left ) . In vitro kinase using Flag - Beclin1 WT mutants ( T38A T119A ) , immunoprecipitated from transfected HeLa , as substrate recombinant ROCK1 was performed . Phosphorylation was detected by 32P - autoradiogram , Flag - Beclin1 were examined by WB .

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(d) Identification of the phosphorylation site on Beclin1. Representation of point mutations in the different domains of Beclin1 are shown (left). In vitro kinase assay using Flag-Beclin1 WT and mutants (T38A and T119A), immunoprecipitated from transfected HeLa cells, as substrate and recombinant ROCK1 was performed. Phosphorylation was detected by 32P-autoradiogram, and Flag-Beclin1 levels were examined by WB.


( e ) Flag - Beclin1 Wt or T119A transfected HeLa were incubated in glucose - rich or nutrient - free ( HBSS ) media , in presence or absence of ROCK1 inhibitor Y27632 . Total extracts were used for IP using Flag agarose . Eluted was analysed by WB against phospho - T119 ( Beclin ) antibody Beclin1 . Input for Beclin1 was run on 7 . 5 % gel immunoblotted .

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(e) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by WB against phospho-T119 (Beclin) antibody and Beclin1. Input for Beclin1 was run on 7.5% gel and immunoblotted.


( a ) Inhibiting ROCK1 activity increases association between Beclin1 Bcl - 2 . HeLa , untreated or treated with 10 μM Y27632 ( top panel ) , were cultured in control or glucose - free ( 4 h ) medium , lysed extracts prepared . Endogenous Bcl - 2 was immunoprecipitated with cross - linked Bcl - 2 agarose resulting eluted immune complexes were resolved by SDS - PAGE blotted against indicated antibodies . EJ shCont . shROCK1 # 1 ( bottom panel ) were grown in control or starvation media for 4 h Bcl - 2 IP was performed as above . Inputs were rerun to confirm ROCK1 knockdown .

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(a) Inhibiting ROCK1 activity increases the association between Beclin1 and Bcl-2. HeLa cells, untreated or treated with 10 μM Y27632 (top panel), were cultured in control or glucose-free (4 h) medium, lysed and cell extracts prepared. Endogenous Bcl-2 was immunoprecipitated with cross-linked Bcl-2 agarose and resulting eluted immune complexes were resolved by SDS-PAGE and blotted against indicated antibodies. EJ shCont. and shROCK1#1 cells (bottom panel) were grown in control or starvation media for 4 h and Bcl-2 IP was performed as above. Inputs were rerun to confirm ROCK1 knockdown.


( b ) HeLa were transfected with full - constructs of Flag - Beclin WT , Flag - Beclin T119A or Flag - Beclin T119E . Flag - Beclin WT were then treated with Y27632 for 8 h . Post transfection treatment with Y27632 , were starved in HBSS medium for 4 h , after which were collected whole lysates prepared . IP with Flag - agarose was performed for 2 h bound to beads were eluted used for western blotting .

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(b) HeLa cells were transfected with full-length constructs of Flag-Beclin WT, Flag-Beclin T119A or Flag-Beclin T119E. Flag-Beclin WT cells were then treated with Y27632 for 8 h. Post transfection and treatment with Y27632, cells were starved in HBSS medium for 4 h, after which cells were collected and whole cell lysates prepared. IP with Flag-agarose was performed for 2 h and proteins bound to beads were eluted and used for western blotting.


( c ) Flag - Beclin1 Wt or T119A transfected HeLa were incubated in glucose - rich or nutrient - free ( HBSS ) media , in presence or absence of ROCK1 inhibitor Y27632 . Total extracts were used for IP using Flag agarose . Eluted was analysed by western blotting against Bcl - 2 Beclin1 . Inputs IP were run on separate gels same exposure is shown .

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(c) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by western blotting against Bcl-2 and Beclin1. Inputs and IP were run on separate gels and same exposure is shown.


( d ) HeLa with a transient knockdown using siCont . siBeclin1 - 3 ′ - untranslated region for 30 h were further left untreated or treated with 10 μM Y27632 . were then incubated in control or HBSS media for 8 h . Whole extracts were prepared , resolved by SDS - PAGE were analysed for Beclin1 , LC3 β - actin .

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(d) HeLa cells with a transient knockdown using siCont. and siBeclin1-3′-untranslated region for 30 h were further left untreated or treated with 10 μM Y27632. Cells were then incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and were analysed for Beclin1, LC3 and β-actin.


( e ) Endogenous LC3 in WT - or T119A - Beclin1 - transfected . HeLa transfected with Flag - Beclin WT ( ± Y27632 ) T119A were cultured in HBSS media for 6 h , fixed with cold acetone endogenous LC3 immunofluorescence was performed . Representative are shown ; scale bar , 20 μm . Arrows indicate punctate LC3 . Graph represents mean ± s . d . , n = 25 , LC3 / .

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(e) Endogenous LC3 staining in WT- or T119A-Beclin1-transfected cells. HeLa cells transfected with Flag-Beclin WT (±Y27632) and T119A were cultured in HBSS media for 6 h, fixed with cold acetone and endogenous LC3 immunofluorescence was performed. Representative images are shown; scale bar, 20 μm. Arrows indicate punctate LC3 staining. Graph represents mean±s.d., n=25, LC3 puncta/cell.


( b ) PCR using tail genomic from three different genotypes , separated on 0 . 7 % agarose gel ( top ) . Western blot of ROCK1 expression in heart tissue from three different genotypes using anti - ROCK1 antibody ( bottom ) . β - Actin was used for loading control in each lane .

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(b) PCR assay using tail genomic DNA from three different genotypes, separated on 0.7% agarose gel (top). Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti-ROCK1 antibody (bottom). β-Actin was used for protein loading control in each lane.


( c ) Mice were fed or starved for 16 h , heart samples were prepared in lysis buffer . Resulting were resolved by SDS - PAGE blotted against ROCK1 , LC3 , autophagy marker p62 β - actin .

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(c) Mice were fed or starved for 16 h, and heart samples were prepared in lysis buffer. Resulting proteins were resolved by SDS-PAGE and blotted against ROCK1, LC3, autophagy marker p62 and β-actin.


( d ) ROCK1 - deficient hearts or control hearts from fed or starved for 16 h were subjected to electron microscopy to identify autophagosome structures . Arrows indicate autophagic vacuoles . Scale bar , 500 nm .

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(d) ROCK1-deficient hearts or control hearts from animals fed or starved for 16 h were subjected to electron microscopy to identify autophagosome structures. Arrows indicate autophagic vacuoles. Scale bar, 500 nm.


( e ) Hearts from fed starved ROCK1 WT KO mice were sectioned , fixed in acetone immunofluorescence was performed against endogenous LC3 . Data are representative of two independent experiments from each genotype . Scale bar , 50 μm .

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(e) Hearts from fed and starved ROCK1 WT and KO mice were sectioned, fixed in acetone and immunofluorescence assay was performed against endogenous LC3. Data are representative of two independent experiments from each genotype. Scale bar, 50 μm.


( E ) Confirmation of thalidomide dependency on six hit using an in vitro binding . Interaction between bls - CRBN FLAG - GST - in presence of DMSO or 50 µM thalidomide was detected using AlphaScreen technology .

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(E) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology.


( F ) In vitro binding for thalidomide , pomalidomide lenalidomide . Interaction between bls - CRBN FLAG - GST - PLZF in presence of DMSO ( 3 . 125 , 6 . 25 , 12 . 5 , 25 , 50 , 100 or 200 µM ) thalidomide , pomalidomide or lenalidomide was analysed using AlphaScreen technology . Data information : relative AlphaScreen ( AS ) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) .

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(F) In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars denote ± standard deviation (n=3).


( E ) In vitro binding ubiquitination of AGIA - PLZF . Empty vector , AGIA - PLZF or FLAG - CRBN expressing HEK293T were lysed lysates were mixed . first immunoprecipitation with anti - AGIA or anti - FLAG antibodies was performed in presence of DMSO or 200 µM lenalidomide . purified AGIA - PLZF or CRL4FLAG - CRBN , including AGIA - PLZF FLAG - CRBN , was incubated with recombinant E1 , E2 HA - ubiquitin in presence of DMSO or 200 µM lenalidomide . second immunoprecipitation was performed using anti - AGIA antibody . Ubiquitination of PLZF was analysed by immunoblot .

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(E) In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide. The purified AGIA-PLZF or CRL4FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2 and HA-ubiquitin in the presence of DMSO or 200 µM lenalidomide. The second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.


( B ) In vitro binding using truncated PLZF . Thalidomide - dependent interaction between bls - CRBN FLAG - GST - PLZF - full ( FL ) or truncated FLAG - GST - PLZF was analysed in presence of DMSO or 50 µM thalidomide using AlphaScreen technology . Data information : relative AlphaScreen ( AS ) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( NS = Not Significant , * * * * P < 0 .

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(B) In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001


( D ) Immunoblot of endogenous SALL4 or PLZF in HuH7 treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . ( E ) Immunoblot of endogenous PLZF or IKZF1 in THP - 1 treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . Data information : Data in bar graphs in ( D ) ( E ) were calculated as relative PLZF band with PLZF band of DMSO as 100 . Error bars donated ± standard deviation ( n = 3 ) P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ) .

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(D) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. (E) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. Data information: Data in bar graphs in (D) and (E) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100. Error bars donated ± standard deviation (n = 3) and P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01 and ***P < 0.001).


( A , B ) Immunoblot of AGIA - MmPLZF ( A ) or - MmSALL4 ( B ) in FLAG - MmCRBN - WT , - MmCRBN - I391V or - MmCRBN - V380E / I391V expressing CRBN - / - HEK293T treated with DMSO or thalidomide ( Tha ) for 16 h . Data information : Data in bar graphs were calculated as relative Mm ) PLZF or SALL4 band with Mm ) PLZF or SALL4 , respectively . band of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) .

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

(A, B) Immunoblot analysis of AGIA-MmPLZF (A) or -MmSALL4 (B) in FLAG-MmCRBN-WT, -MmCRBN-I391V or -MmCRBN-V380E/I391V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. Data information: Data in bar graphs were calculated as relative Mm) PLZF or SALL4 band intensity with Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


( C , D ) Immunoblot of AGIA - GgPLZF ( C ) or - GgSALL4 ( D ) in FLAG - GgCRBN - WT or - GgCRBN - I390V expressing CRBN - / - HEK293T treated with DMSO or thalidomide ( Tha ) for 16 h . ( E , F ) Immunoblot of AGIA - Mm PLZF ( E ) or - MmSALL4 ( F ) in FLAG - MmCRBN - WT expressing CRBN - / - HEK293T treated with indicated of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . ( G , H ) Immunoblot of AGIA - GgPLZF ( G ) or - GgSALL4 ( H ) in FLAG - GgCRBN - WT expressing CRBN - / - HEK293T treated with indicated of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . Data information : Data in bar graphs were calculated as relative ( Gg or Mm ) PLZF or SALL4 band with ( Gg or Mm ) PLZF or SALL4 , respectively . band of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) .

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

(C, D) Immunoblot analysis of AGIA-GgPLZF (C) or -GgSALL4 (D) in FLAG-GgCRBN-WT or -GgCRBN-I390V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. (E, F) Immunoblot analysis of AGIA-Mm PLZF (E) or -MmSALL4 (F) in FLAG-MmCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. (G, H) Immunoblot analysis of AGIA-GgPLZF (G) or -GgSALL4 (H) in FLAG-GgCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. Data information: Data in bar graphs were calculated as relative (Gg or Mm) PLZF or SALL4 band intensity with (Gg or Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


( E ) Skeletal patterning of forelimb hindlimb in E6 chicken infected RCAN packaging shControl ( n = 10 ) or shPLZF ( n = 9 ) were analysed by Victoria blue . h ; humerus , r ; radius , u ; ulna , fe ; femur , fi ; fibula , t ; tibia . Data in bar graphs were calculated as relative bone with bone of shControl as 100 . Error bars denote ± standard deviation ( forelimb hindlimb shControl ( n = 3 ) , forelimb shPLZF ( n = 5 ) or hindlimb shPLZF ( n = 6 ) ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( * * P < 0 . 01 * * * * P < 0 . 0001 ) .

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

(E) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl (n = 3), forelimb shPLZF (n = 5) or hindlimb shPLZF (n = 6)). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01 and ****P < 0.0001).


( F ) show E4 chicken treated with HBC ( control , n = 10 , C1 , C3 , C5 C6 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 , T1 , T2 , T6 T9 ) or 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 , H1 , H2 , H8 H9 ) corresponding to immunoblot in Fig 7G . Red arrows show treated regions . Data information Data in bar graphs in ( F ) were calculated as relative limb bud with left limb bud as one . Error bars denote ± standard deviation ( F ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( NS = Not Significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Photographs show E4 chicken embryos treated with HBC (control, n = 10, C1, C3, C5 and C6), 1 µg/µl (3.9 mM) thalidomide (n = 11, T1, T2, T6 and T9) or 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n = 10, H1, H2, H8 and H9) corresponding to immunoblot analysis in Fig 7G. Red arrows show treated regions. Data information Data in bar graphs in (F) were calculated as relative limb bud length with left limb bud length as one. Error bars denote ± standard deviation (F), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, **P < 0.01, and ***P < 0.001).


( G ) Immunoblot of PLZF or SALL4 from tissue of E4 chicken forelimb bud . Endogenous PLZF or SALL4 expression was detected by immunoblot using chicken treated with HBC ( n = 10 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 ) 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 ) . Data information : Data in bar graphs in ( G ) were calculated as relative GgPLZF or GgSALL4 band with GgPLZF or GgSALL4 , respectively . band of HBC was 100 . Error bars denote ± standard deviation ( ( E ) , n = 3 or ( G ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 ) .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud. Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC (n =10), 1 µg/µl (3.9 mM) thalidomide (n =11) and 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n =10). Data information: Data in bar graphs in (G) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4, respectively. The band intensity of HBC was 100. Error bars denote ± standard deviation ((E), n = 3 or (G), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, and **P < 0.01).


E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS - GgPLZF was treated with HBC or 1 µg / µl ( 3 . 9 mM ) thalidomide , chicken forelimb bud were observed in bright field ( BF ) or of enhanced GFP ( EGFP ) . Fgf10 / Fgf8 expression was analysed by section by in situ hybridisation . ( Q ) Quantitative of of chicken limb bud Data in bar graphs in ( Q ) were calculated as relative limb bud with limb bud of control as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( * * P < 0 . 01 ) .

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

E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS-GgPLZF was treated with HBC or 1 µg/µl (3.9 mM) thalidomide, and the chicken forelimb bud phenotypes were observed in bright field (BF) or fluorescence of enhanced GFP (EGFP). Fgf10/Fgf8 expression was analysed by section by in situ hybridisation. (Q) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in (Q) were calculated as relative limb bud length with limb bud length of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01).


( R ) Quantitative of Fgf10 / Fgf8 expression Data in bar graphs in ( R ) were calculated as relative Fgf10 / Fgf8 mRNA expressions with Fgf10 / Fgf8 mRNA expressions of control as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc ( * P < 0 . 05 * * P < 0 . 01 ) .

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

(R) Quantitative analysis of Fgf10/Fgf8 expression Data in bar graphs in (R) were calculated as relative Fgf10/Fgf8 mRNA expressions with Fgf10/Fgf8 mRNA expressions of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (*P < 0.05 and **P < 0.01).


Decreased Drp1S616 phosphorylation in PINK1 - null HEK293 Lysates of PINK1 - null ( PINK1KO ) parkin - null ( parkinKO ) HEK293 ( a ) their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Two independent PINK1KO parkinKO HEK293 lines ( 1 , 2 ) were shown . Student ' s . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

Decreased Drp1S616 phosphorylation in PINK1-null HEK293 cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) HEK293 cells (a) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO HEK293 cell lines (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Decreased Drp1S616 phosphorylation in PINK1 - null MEF Lysates of PINK1 - null ( PINK1KO ) parkin - null ( parkinKO ) MEF ( c ) their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Student ' s . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

Decreased Drp1S616 phosphorylation in PINK1-null MEF cells Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) MEF cells (c) and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


Decreased Drp1S616 phosphorylation in PINK1 - null mouse substantial nigra tissues . Lysates of PINK1 - null ( PINK1KO ) parkin - null ( parkinKO ) mouse substantial nigra tissues ( e ) , their matched wildtype controls ( WT ) were immuno - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho ( Ser637 ) - Drp1 ( pS637 ) or Drp1 . β - actin was detected as a loading control . Quantitation of pS616 / Drp1 for each experiment is shown Two independent PINK1KO parkinKO substantial nigra from two separate PINK1KO parkinKO mice ( 1 , 2 ) were shown . Student ' s . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

Decreased Drp1S616 phosphorylation in PINK1-null mouse substantial nigra tissues. Lysates of PINK1-null (PINK1KO) and parkin-null (parkinKO) mouse substantial nigra tissues (e), and their matched wildtype controls (WT) were immuno-detected with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho (Ser637)-Drp1 (pS637) or Drp1. β-actin was detected as a loading control. Quantitation of pS616/Drp1 for each experiment is shown Two independent PINK1KO and parkinKO substantial nigra from two separate PINK1KO and parkinKO mice (1, 2) were shown. Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( g , h ) . Overexpression of PINK1 , not PINK1 kinase mutants , reverses Drp1S616 phosphorylation in PINK1KO HEK293 . Lysates of PINK1KO PINK1WT control ( WT ) HEK293 expressing PINK1 variants were immuno - detected with an anti - phospho ( Ser616 ) - Drp1 antibody ( pS616 ) , an anti - Drp1 antibody ( Drp1 ) an anti - PINK1 antibody ( PINK1 ) . with mock transfection ( Blank ) transfected with an empty plasmid ( 3 . 1 ) were included as controls ( g ) . Quantitation of pS616 / Drp1 is shown ( h ) . PINK1 : wildtype PINK1 ; D384N : PINK1 kinase - dead mutant ; G309D , pathogenic PINK1 mutant ; ∆ 110 : PINK1 mito - target deficient mutant . Student ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

(g, h). Overexpression of PINK1, not PINK1 kinase mutants, reverses Drp1S616 phosphorylation in PINK1KO HEK293 cells. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells expressing PINK1 variants were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1) and an anti-PINK1 antibody (PINK1). Cells with mock transfection (Blank) and transfected with an empty plasmid (3.1) were included as controls (g). Quantitation of pS616/Drp1 is shown (h). PINK1: wildtype PINK1; D384N: PINK1 kinase-dead mutant; G309D, pathogenic PINK1 mutant; ∆110: PINK1 mito-target deficient mutant. Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( i , j ) . PINK1 deficiency does not affect expression kinase activity of CDK1 ERK1 / 2 . Lysates of PINK1KO PINK1WT control ( WT ) HEK293 were immuno - detected with an anti - phospho ( Ser616 ) - Drp1 antibody ( pS616 ) , an anti - Drp1 antibody ( Drp1 ) , an anti - PINK1 antibody ( PINK1 ) , an anti - CDK1 antibody ( CDK1 ) , an anti - p44 / p42 MAPK antibody ( ERK1 / 2 ) , an anti - phospho ( Thr161 ) - CDK1 antibody ( pThr161 ) , an anti - phospho ( Thr202 / 204 ) - p44 / p42 MAPK antibody ( pThr202 / 204 ) . β - actin was detected as a loading control ( i ) . Quantitation of CDK1Thr161 phosphorylation ( pThr161 ) ERK1 / 2Thr202 / 204 phosphorylation ( pThr202 / 204 ) was shown ( j ) . Student ' s . ns : no significance . Data was presented as mean ± of three independent experiments .

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

(i, j). PINK1 deficiency does not affect expression and kinase activity of CDK1 and ERK1/2. Lysates of PINK1KO and PINK1WT control (WT) HEK293 cells were immuno-detected with an anti-phospho(Ser616)-Drp1 antibody (pS616), an anti-Drp1 antibody (Drp1), an anti-PINK1 antibody (PINK1), an anti-CDK1 antibody(CDK1), an anti-p44/p42 MAPK antibody (ERK1/2), an anti-phospho(Thr161)-CDK1 antibody (pThr161), and an anti-phospho(Thr202/204)-p44/p42 MAPK antibody (pThr202/204). β-actin was detected as a loading control (i). Quantitation of CDK1Thr161 phosphorylation (pThr161) and ERK1/2Thr202/204 phosphorylation (pThr202/204) was shown (j). Student's test. ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( l , m ) . PINK1 phosphorylates Drp1S616 in vitro . Recombinant TcPINK1 was incubated with either GST - Drp1518 - 736 ( WT ) or GST - Drp1518 - 736 , S616A ( S616A ) in presence ( + ) / absence ( - ) of ATP . After heat inactivation , reactions were treated with ( + ) / without ( - ) shrimp alkaline phosphatase ( SAP ) . Phosphorylation of Drp1S616 ( pS616 ) was immuno - detected ( l , upper panel ) . Total Drp1 ( l , middle panel ) loading stained with Coomassie blue ( l , lower panel ) were shown . Quantitation of pS616 / Drp1 was shown ( m ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 . Data was presented as mean ± of three independent experiments .

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

(l, m). PINK1 phosphorylates Drp1S616 in vitro. Recombinant TcPINK1 was incubated with either GST-Drp1518-736 (WT) or GST-Drp1518-736, S616A (S616A) in the presence (+)/absence (-) of ATP. After heat inactivation, reactions were treated with (+)/without (-) shrimp alkaline phosphatase (SAP). Phosphorylation of Drp1S616 (pS616) was immuno-detected (l, upper panel). Total Drp1 (l, middle panel) and protein loading stained with Coomassie blue staining (l, lower panel) were shown. Quantitation of pS616/Drp1 ratio was shown (m). One-way ANOVA followed with Tukey's test. ***p<0.001. Data was presented as mean ± SEM of three independent experiments.


( n , o ) . Label - free relative quantitative mass spectrum of Drp1S616 phosphorylation . Representative spectra for Drp1 phospho - peptide ( S616 ) SKPIPMPA ( p ) SPQK non - phospho - peptide SKPIPMPA ( p ) SPQK . Spectra obtained for peptides from PINK1WT HEK293 are shown ( n ) . relative ratios of phospho - peptides containing phosphor - Ser616 to peptides containing Ser616 from PINK1WT PINK1KO HEK293 samples were calculated plotted ( 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 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 I-CONTROLLED_VAR O O O O O O O O O

(n, o). Label-free relative quantitative mass spectrum analysis of Drp1S616 phosphorylation. Representative spectra for the Drp1 phospho-peptide (S616) SKPIPMPA(p)SPQK and non-phospho-peptide SKPIPMPA(p)SPQK. Spectra obtained for peptides from PINK1WT HEK293 cells are shown (n). The relative ratios of the phospho-peptides containing phosphor-Ser616 to peptides containing Ser616 from PINK1WT and PINK1KO HEK293 samples were calculated and plotted (o).


( p , q ) . Km Kcat for PINK1 - mediated phosphorylation of ubiquitin Drp1 . In vitro kinase assays were performed with different concentrations of ubiquitin ( p ) or Drp1 ( q ) with TcPINK1 at fixed ATP ( 500 μM ) . Results were analyzed on phos ‐ tag gels modeled to Michaelis - Menten equation . given graphs represent global fits to data collected from 3 sets of reactions for both Ub Drp1 performed independently . Km : michaelis constant . Kcat : catalytic constant . Bars represent mean ± .

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

(p, q). Km and Kcat for PINK1-mediated phosphorylation of ubiquitin and Drp1. In vitro kinase assays were performed with different concentrations of ubiquitin (p) or Drp1 (q) with TcPINK1 at fixed ATP concentration (500 μM). Results were analyzed on phos‐tag gels and modeled to the Michaelis-Menten equation. The given graphs represent global fits to data collected from 3 sets of reactions for both Ub and Drp1 performed independently. Km: michaelis constant. Kcat: catalytic constant. Bars represent the mean ± SEM.


( a , b ) . Decreased Drp1S616 phosphorylation in PINK1 - null neurons . Lysates of DIV6 neurons generated from PINK1KO PINK1WT control mice were immune - detected with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) or Drp1 . Tuj1 was detected as a loading control ( a ) . Quantitation of pS616 / Drp1 for each experiment is shown ( b ) . Student ' s . * * p < 0 . 01 . Data was presented as mean ± of three independent experiments .

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

(a, b). Decreased Drp1S616 phosphorylation in PINK1-null primary neurons. Lysates of DIV6 neurons generated from PINK1KO and PINK1WT control mice were immune-detected with antibodies against either phospho (Ser616)-Drp1 (pS616) or Drp1. Tuj1 was detected as a loading control (a). Quantitation of pS616/Drp1 for each experiment is shown (b). Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.


( c , d ) . of mitochondria in mouse substantial nigra . Sections of substantial nigra from 18 - month - old PINK1KO PINK1WT control mice were analyzed under ( c , left panels , scale bar = 2 µm ) . Amplified of mitochondria from dot frames gated region are shown ( c , right panels , scale bar = 1 µm ) . Asterisks indicate abnormal elongated mitochondria . A quantitative of mitochondria with perimeter > 2µm is shown ( d ) . Over 300 identifiable mitochondria ( cristae / or double ) per mice were randomly blindly selected for perimeter measurement . Student ' s . * p < 0 . 05 . Data was presented as mean ± of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). TEM analysis of mitochondria in mouse substantial nigra. Sections of substantial nigra from 18-month-old PINK1KO and PINK1WT control mice were analyzed under TEM (c, left panels, scale bar=2 µm). Amplified images of mitochondria from dot line frames gated region are shown (c, right panels, scale bar=1 µm). Asterisks indicate abnormal elongated mitochondria. A quantitative analysis of mitochondria with perimeter >2µm is shown (d). Over 300 identifiable mitochondria (cristae and/or double membrane) per mice were randomly and blindly selected for perimeter measurement. Student's test. *p<0.05. Data was presented as mean ± SEM of three independent experiments.


( e , f ) . Overexpression of Drp1WT , but not phosphorylation mutant Drp1S616A , suppresses mitochondrial fusion in dendrites of PINK1KO neurons . ( e ) . DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co - transfected mito - GFP with either pcDNA3 . 1 ( 3 . 1 ) or Drp1WT or Drp1S616A . Neurons were immuno - detected with an antibody against myc - tag ( Myc ) ( red , to detect exogenous Drp1 ) . Mito - GFP : green , to detect mitochondria . Representative were shown , scale bar = 25 μm . Magnified dendritic shafts gated by a dot frame are shown ( Zoom , right panels , scale bar = 5 μm ) . ( f ) . of Mito - index in dendrites of neurons from experiments of ( e ) . 10 neurons were randomly selected analyzed in each experiment . One - way ANOVA followed with Tukey ' s . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

(e, f). Overexpression of Drp1WT, but not phosphorylation mutant Drp1S616A, suppresses mitochondrial fusion in dendrites of PINK1KO primary neurons. (e). DIV4 neurons generated from PINK1 WT control or PINK1KO mice were co-transfected mito-GFP with either pcDNA3.1 (3.1) or Drp1WT or Drp1S616A. Neurons were immuno-detected with an antibody against myc-tag (Myc) (red, to detect exogenous Drp1). Mito-GFP: green, to detect mitochondria. Representative images were shown, scale bar=25 μm. Magnified dendritic shafts gated by a dot line frame are shown (Zoom, right panels, scale bar=5 μm).(f). Quantification of Mito-index in dendrites of neurons from experiments of (e). 10 neurons were randomly selected and analyzed in each experiment. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - c ) . Drp1 is required for mitochondrial fission induced by PINK1 . Drp1 - null HeLa ( Drp1KO ) their wildtype controls ( Drp1WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP with FRB - MTS . were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( Δ110 - PINK1 , green ) . Nuclei were labeled with DAPI ( blue ) . treated with solvent ( DMSO ) were used as a treatment control . Representative were shown ( a , upper panel , scale bar = 25 μm ) . Higher magnification are also included ( a , lower panel , scale bar = 10 μm ) . Mitochondrial in different transfections was quantified . ( b , c ) . Student ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

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

(a-c). Drp1 is required for mitochondrial fission induced by PINK1. Drp1-null HeLa cells (Drp1KO) and their wildtype controls (Drp1WT) were co-transfected Δ110-PINK1-GFP-FKBP with FRB-MTS. Cells were induced with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (Δ110-PINK1, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown (a, upper panel, scale bar=25 μm). Higher magnification images are also included (a, lower panel, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified. (b, c). Student's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( d - f ) . Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1 . Drp1KO HeLa were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A . were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , blue ) , exogenous Drp1 ( myc , red ) , Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . treated with solvent ( DMSO ) were used as a treatment control . Representative were shown , scale bar = 25 μm ( d ) . Higher magnification are also included ( d , Zoom , scale bar = 5 μm ) . Mitochondrial in different transfections was quantified ( e , f ) . Student ' s . * * p < 0 . 01 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

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

(d-f). Drp1S616 phosphorylation is required for mitochondrial fission induced by PINK1. Drp1KO HeLa cells were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either a plasmid encoding Drp1WT or a Drp1 dephosphorylation mimic mutant Drp1S616A. Cells were treated with 250 nM rapalog for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, blue), exogenous Drp1 (myc, red), Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm (d). Higher magnification images are also included (d, Zoom, scale bar=5 μm). Mitochondrial morphology in different transfections was quantified (e, f). Student's test. **p<0.01, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( g , h ) . Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616 . HEK293 co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP were treated with 250 nM rapalog ( Rapalog ) . After 2hours treatment , cytosol protion ( cyto ) mitochondria ( Mito ) were fractionated immunobloted with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , Drp1 ( Drp1 ) , or GFP ( PINK1 ) . Tom20 β - actin were detected as mitochondrial loading control cytosol loading control , respectively ( g ) . Quantitation of pS616 / Drp1 in various conditions was shown ( h ) . Student ' s . * * p < 0 . 01 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

(g, h). Targeting PINK1 kinase domain to OMM increases mitochondrial localization of phosphorylated Drp1S616. HEK293 cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP were treated with 250 nM rapalog (Rapalog). After 2hours treatment, cytosol protion (cyto) and mitochondria (Mito) were fractionated and immunobloted with antibodies against either phospho (Ser616)-Drp1 (pS616), Drp1 (Drp1), or GFP (PINK1). Tom20 and β-actin were detected as mitochondrial loading control and cytosol loading control, respectively (g). Quantitation of pS616/Drp1 in various conditions was shown (h). Student's test. **p<0.01, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( i , j ) . Targeting PINK1 kinase domain to OMM increases mitochondrial localization of Drp1 . HeLa co - transfected FRB - MTS with Δ110 - PINK1 - GFP - FKBP ( FKBP - PINK1 ) were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . treated with solvent ( DMSO ) were included as a control . were immunostained with antibodies against either Tom20 , Drp1 GFP ( PINK1 - FKBP ) . Representative were shown , scale bar = 2 μm ( i ) . Higher magnification from gated box are shown ( bottom panels ) . Arrows indicate Drp1 . Mitochondria - associated ( yellow ) non - associated Drp1 ( Red ) are shown , scale bar = 1 μm . Mitochondria - associated Drp1 were analyzed by using an ImageJ plugin - Coloc 2 expressed as Manders ' Colocalization Coefficients ( MCC ) ( j ) . Three different regions from each > 15 for each experimental group were analyzed . Student ' s . * * p < 0 . 01 . Data was presented as mean ± of three independent experiments .

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(i, j). Targeting PINK1 kinase domain to the OMM increases mitochondrial localization of Drp1. HeLa cells co-transfected FRB-MTS with Δ110-PINK1-GFP-FKBP (FKBP-PINK1) were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cells were immunostained with antibodies against either Tom20, Drp1 and GFP (PINK1-FKBP). Representative images were shown, scale bar=2 μm (i). Higher magnification images from gated box are shown (bottom panels). Arrows indicate Drp1 puncta. Mitochondria-associated (yellow) and non-associated Drp1(Red) are shown, scale bar=1 μm. Mitochondria-associated Drp1 puncta were analyzed by using an ImageJ plugin-Coloc 2 and expressed as the Manders' Colocalization Coefficients (MCC) (j). Three different regions from each cell and >15 cells for each experimental group were analyzed. Student's test. **p<0.01. Data was presented as mean ± SEM of three independent experiments.


( k , l ) . PINK1 regulates S616 S637 phosphorylation of Drp1 via independent mechanisms . DRP1KO HEK293 co - transfected FRB - MTS / Δ110 - PINK1 - GFP - FKBP ( PINK1WT ) with either myc - Drp1WT or myc - Drp1S616A were treated with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase . treated with solvent ( DMSO ) were included as a control . lysates were immunoblotting with antibodies against either phospho ( Ser616 ) - Drp1 ( pS616 ) , phospho - ( Ser637 ) - Drp1 ( pS637 ) , Drp1 GFP ( to detect PINK1 - FKBP ) ( k ) . β - actin was detected as a loading control . Quantitation of pS637 / Drp1 in various conditions was shown ( l ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments .

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

(k, l). PINK1 regulates S616 and S637 phosphorylation of Drp1 via independent mechanisms. DRP1KO HEK293 cells co-transfected FRB-MTS/Δ110-PINK1-GFP-FKBP (PINK1WT) with either myc-Drp1WT or myc-Drp1S616A were treated with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase. Cells treated with solvent (DMSO) were included as a control. Cell lysates were immunoblotting with antibodies against either phospho (Ser616)-Drp1 (pS616), phospho-(Ser637)-Drp1 (pS637), Drp1 and GFP (to detect PINK1-FKBP) (k). β-actin was detected as a loading control. Quantitation of pS637/Drp1 in various conditions was shown (l). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments.


( a - e ) . ATG5 is dispensable for PINK1 - mediated mitochondrial fission . MEF derived from ATG5 - null ( ATG5KO ) their wildtype control mice ( ATG5WT ) were co - transfected Δ110 - PINK1 - GFP - FKBP / FRB - MTS with either PINK1WT or a kinase - dead mutant PINK1D384N . were induced with 250 nM rapalog ( Rapalog ) for 2 hours to activate PINK1 kinase , followed by immunodetecting mitochondria ( TOM20 , red ) Δ110 - PINK1 - GFP - FKBP / FRB - MTS ( PINK1 - FKBP , green ) . treated with solvent ( DMSO ) were used as a treatment control . Representative are shown , scale bar = 25 μm ( a ) . Higher magnification are also included ( panels beneath large , scale bar = 10 μm ) . Mitochondrial in different transfections was quantified ( b - e ) . Student ' s . * p < 0 . 05 , * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

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

(a-e). ATG5 is dispensable for PINK1-mediated mitochondrial fission. MEF cells derived from ATG5-null (ATG5KO) and their wildtype control mice (ATG5WT) were co-transfected Δ110-PINK1-GFP-FKBP /FRB-MTS with either PINK1WT or a kinase-dead mutant PINK1D384N. Cells were induced with 250 nM rapalog (Rapalog) for 2 hours to activate PINK1 kinase, followed by immunodetecting mitochondria (TOM20, red) and Δ110-PINK1-GFP-FKBP /FRB-MTS (PINK1-FKBP, green). Cells treated with solvent (DMSO) were used as a treatment control. Representative images are shown, scale bar=25 μm (a). Higher magnification images are also included (panels beneath the large cell images, scale bar=10 μm). Mitochondrial morphology in different transfections was quantified (b-e). Student's test. *p<0.05, ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT Drp1KO HeLa were transfected with GFP - parkin . were treated with 20 μM CCCP for 2 hours ( f ) followed by immunodetecting mitochondria ( TOM20 , red ) parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . treated with solvent ( DMSO ) were used as a treatment control . Representative were shown , scale bar = 25 μm . with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

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(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 20 μM CCCP for 2 hours (f) followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.


( f - i ) . Drp1 is dispensable for recruitment of parkin to mitochondria . Drp1WT Drp1KO HeLa were transfected with GFP - parkin . were treated with 150 μM actinonin for 6 hours ( h ) , followed by immunodetecting mitochondria ( TOM20 , red ) parkin ( GFP - parkin , green ) . Nuclei were labeled with DAPI ( blue ) . treated with solvent ( DMSO ) were used as a treatment control . Representative were shown , scale bar = 25 μm . with mitochondrial parkin ( % ) in different experimental group was quantified ( g , i ) . One - way ANOVA followed with Tukey ' s . * * * p < 0 . 001 , ns : no significance . Data was presented as mean ± of three independent experiments . For each condition , > 100 were analyzed .

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

(f-i). Drp1 is dispensable for recruitment of parkin to mitochondria. Drp1WT and Drp1KO HeLa cells were transfected with GFP-parkin. Cells were treated with 150 μM actinonin for 6 hours (h), followed by immunodetecting mitochondria (TOM20, red) and parkin (GFP-parkin, green). Nuclei were labeled with DAPI (blue). Cells treated with solvent (DMSO) were used as a treatment control. Representative images were shown, scale bar=25 μm. Cell with mitochondrial parkin (%) in different experimental group was quantified (g, i). One-way ANOVA followed with Tukey's test. ***p<0.001, ns: no significance. Data was presented as mean ± SEM of three independent experiments. For each condition, >100 cells were analyzed.