Figure 3A - R . Histopathological analysis of the brain in Tha - RABV - infected non - treated mice ( at 11 dpi ) , versus surviving mice ( at 100 dpi ) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail . The detection of Tha - RABV , using immunohistochemistry ( IHC ) on brain sections , revealed a heterogeneous invasion between the non - treated mice , ranging from extensive ( A ) to multifocal ( B ) . Higher magnification of extensive invasion in the cortex ( F ) , hippocampus ( G ) , brain stem ( H ) and cerebellum ( I ) . ( C , J , K , L , M ) In contrast , even if a non - specific IHC signal was sometimes observed , no virus was detected on the brain sections of treated mice at 100 days post - infection . Only rare signs of neuro - inflammation were observed , as perivascular cuffing ( D ) ( 2 / 7 mice ) and minimal meningitis ( E ) ( 3 / 7 mice ) . Data information : A - C and F - M : Tha - RABV IHC D - E : HE staining . F - I are high magnifications of A ; J - M are high magnifications of C ( N , O ) Microglial cells were in general not reactive , but ( P ) small foci of reactive microglia ( hyperplasia with thicker cell processes ) could randomly be detected . As for control non - treated mice , reactive microglial cells ( Q ) ( 2 / 7 mice ) or resting microglia ( R ) were observed in treated mice . Representative images . Data information : N - R : Iba1 IHC to detect microglial cells O - P are high magnifications of N .

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

Figure 3A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). Data information: A-C and F-M: Tha-RABV IHC D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C(N,O) Microglial cells were in general not reactive, but (P) small foci of reactive microglia (hyperplasia with thicker cell processes) could randomly be detected. As for control non-treated mice, reactive microglial cells (Q) (2/7 mice) or resting microglia (R) were observed in treated mice. Representative images. Data information: N-R: Iba1 IHC to detect microglial cells O-P are high magnifications of N.


Figure 1 A Western blot analysis of LC3 in SH - SY5Y cells after IU1 treatment ( 100 μM , 24h ) . Graphs represent mean ± SEM . N = 8 B - D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control ( DMSO ) or IU1 treated cells . At least 55 cells were analysed from 3 different experiments . Graphs represent mean ± SEM E Western blot analysis of mitochondrial marker proteins ATP5a , HSP60 and normalised by actin after 24h of DMSO ( C ) or IU1 ( 100 μM ) treatment . N = 3 . Graphs represent mean ± SEM 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 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 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

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

Figure 1 A Western blot analysis of LC3 in SH-SY5Y cells after IU1 treatment (100 μM, 24h). Graphs represent mean± SEM. N= 8  B-D Electron microscope images were taken to quantify the number of autophagosomes and autolysosome present in control (DMSO) or IU1 treated cells. At least 55 cells were analysed from 3 different experiments. Graphs represent mean± SEM  E Western blot analysis of mitochondrial marker proteins ATP5a, HSP60 and normalised by actin after 24h of DMSO (C) or IU1 (100 μM) treatment. N=3. Graphs represent mean± SEM  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 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 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


Figure 2 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 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 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 ;

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

Figure 2 A-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 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 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;


Figure 3 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 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

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

Figure 3 A-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 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


Figure 4 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 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 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

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

Figure 4 A-B 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 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 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


Figure 5Electron 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 countedRepresentative 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 experimentsElectron 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 countedRepresentative 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 experimentsElectron 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 countedRepresentative 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 experimentsElectron 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 experimentsRepresentative 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

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

Figure 5Electron 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 countedRepresentative 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 experimentsElectron 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 countedRepresentative 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 experimentsElectron 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 countedRepresentative 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 experimentsElectron 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 experimentsRepresentative 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


Figure 6 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 ± SEM of the climbing performance of flies of the indicated genotypes . n = 4 - 7 . ANOVA followed by Newman Keuls test 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 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 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

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

Figure 6 A 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± SEM of the climbing performance of flies of the indicated genotypes. n=4-7. ANOVA followed by Newman Keuls test  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 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 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


Figure 7 ( 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 ± SEM of the climbing performance of flies of the indicated genotypes . One way ANOVA , Newman Keuls test , n = 3 ( 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 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

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

Figure 7 (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± SEM of the climbing performance of flies of the indicated genotypes. One way ANOVA, Newman Keuls test, n=3  (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 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


f1 ( 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 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 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 .

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

f1(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 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 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.


f2 ( 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 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 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 . 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 . ) 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 ) .

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

f2(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 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 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. 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.) 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).


f3 ( 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 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 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 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 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 .

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

f3(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 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 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 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 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.


f4 ( 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 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 . 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 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 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 .

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

f4(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 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. 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 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 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.


f5 ( 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 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 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 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 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 .

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

f5(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 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 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 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 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.


f6 ( 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 , 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 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 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 .

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

f6(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, 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 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 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.


Figure 1 ( 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 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 ) .

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

Figure 1(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 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).


Figure 2 ( 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 .

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

Figure 2(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.


Figure 3 ( 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 .

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

Figure 3(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


Figure 4 ( 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 ) .

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

Figure 4(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).


Figure 5 ( 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 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 ) .

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

Figure 5(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 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).


Figure 6 ( 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 ) .

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

Figure 6(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).


Figure 7 ( 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 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 ) .

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

Figure 7(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 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).


Figure 8E3 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 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 ) .

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

Figure 8E3 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 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).


Figure 1Decreased 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 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 ) 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 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 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 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 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 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 .

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

Figure 1Decreased 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 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) 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 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 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 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 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 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.


Figure 2 ( 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 ) . 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 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 .

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

Figure 2(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). 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 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.