A . Representative pictures of double immunostaining with VEcad ( red ; left middle panel ) KLF4 ( green , left panel ) or TCF4 ( green , middle panel ) , with CD31 ( red ; right panels , confocal pictures ) TCF4 ( , right panel ) on Control CriptoMy - LOF TA sections at day 5 after injury . B - D . of VEcad / KLF4 ( B ) , VEcad / TCF4 ( C ) CD31 / TCF4 ( D ) double positive per ( mm2 ) in Control CriptoMy - LOF TA sections at day 5 after injury . Data information : Nuclei were counterstained with DAPI ( blue ) .

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A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury. B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury. Data information: Nuclei were counterstained with DAPI (blue).


E . Representative Confocal pictures of CD31 + ( red ) PDGFRα + ( green ) immunostaining on Control CriptoMy - LOF TA sections at day 5 after injury . F - G . of CD31 + / PDGFRα + ( F ) CD31 - / PDGFRα + ( G ) per ( mm2 ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury. F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). Data information: Nuclei were counterstained with DAPI (blue).


H - I . Representative pictures of triple immunostaining with VEcad ( red ) , KLF4 ( green ) pSMAD3 ( ) on Control CriptoMy - LOF TA sections at day 5 after injury ( H ) of VEcad / KLF4 / pSMAD3 triple positive per ( mm2 ; I ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I). Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) KLF4 ( green ) on Control CriptoMy - LOF TA sections at day 5 after re - injury ( CTX - II Data information : Nuclei were counterstained with DAPI ( blue ) .

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Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II Data information: Nuclei were counterstained with DAPI (blue).


Representative pictures of double immunostaining with VEcad ( red ) KLF4 ( green ) on diaphragm sections of mdx - Control mdx - CriptoMy - LOF Data information : Nuclei were counterstained with DAPI ( blue ) .

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Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF Data information: Nuclei were counterstained with DAPI (blue).


of VEcad / KLF4 double positive per ( mm2 ) in Control CriptoMy - LOF TA sections at day 5 after re - injury ( CTX -

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Quantification of VEcad/KLF4 double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II


of VEcad / KLF4 double positive per ( mm2 ) in diaphragm sections of mdx - Control mdx - CriptoMy -

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Quantification of VEcad/KLF4 double positive cells per area (mm2) in diaphragm sections of mdx-Control and mdx-CriptoMy-LOF


A ROS were measured by flow cytometry after with specific oxidative stress detection dyes ( CellROX green , Molecular Probes , USA ) in HC ( n = 4 ) DM ( n = 4 ) platelets . ( * * p = 0 . 032 vs . HC ) .

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A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).


B Western blot of ROS downstream signaling molecules p53 phosphorylated p53 in HC ( # 1 - 3 ) DM ( # 1 - 8 ) platelets . on HC ( n = 5 ) DM ( n = 18 ) individuals . ( * * p = 0 . 0025 vs . HC ) . GAPDH served as loading control .

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B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.


C immunostaining for phosphorylated p53 in HC DM platelets . Graph indicates HC DM platelets positive for phosphorylated p53 signal . y - axis indicates of phosphorylated p53 positive from total ( * * p = 0 . 0064 vs . HC ) . Representative figure from n = 5 .

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C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5.


D Mitochondrial potential ( ΔΨm ) was measured by MitoTracker ( 50nM for 15 min ) in HC DM platelets . Graph indicates signal of MitoTracker in HC DM platelets . y - axis indicates fold of signal in HC DM platelet ( * * p = 1 . 1608E - 05 vs HC ) . Signal of each group was converted to fold change compared with HC values . Representative figure from HC ( n = 6 ) DM ( n = 4 ) .

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D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4).


E ΔΨm platelet apoptosis were measured by flow cytometry . ΔΨm was detected using TMRM apoptosis was assessed with Annexin - V ( PS externalization ) . Representative figure from n = 3 .

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E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.


F Western blot of apoptosis - related pp53 , p53 , cytochrome c , active caspase 3 in HC DM platelets . Tubulin was used as loading control . Representative figure from n = 3 .

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F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3.


G Western blot showing release of cytochrome c ( Cyto c ) into cytosol in DM . Representative figure from n = 3 .

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G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3.


H EM of platelets mitochondria from HC DM demonstrating examples of mitochondria damage in DM platelets ( DM ) compared to a typical healthy mitochondria ( HC ) . Arrows indicate disrupted outer membranes . I of mitochondria in HC ( n = 33 ) DM ( n = 17 ) platelets . y - axis indicates average number of mitochondria in HC DM single platelet EM view ( * * p = 5 . 50811E - 05 vs . HC ) . J of healthy damaged mitochondria in HC DM platelets .

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H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.


A Western blot of autophagy markers , Beclin1 , ATG3 , ATG7 , ATG12 - 5 , LC3I / II , mitophagy - related Parkin PINK1 in HC ( # 1 - 3 ) DM platelets ( # 1 - 8 ) . B of autophagy markers mitophagy - related from HC ( # 1 - 5 ) DM ( # 1 - 18 ) individuals . ( Beclin1 ; * * p = 0 . 0003 , ATG12 ; * p = 0 . 0388 , ATG3 ; * p = 0 . 0213 , ATG7 ; p = 0 . 4051 , LC3 ; * p = 0 . 0298 , PINK1 ; * p = 0 . 0155 , Parkin ; * p = 0 . 0103 vs . HC ) . GAPDH served as a loading control .

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A Western blot analysis of autophagy markers, Beclin1, ATG3, ATG7, ATG12-5, and LC3I/II, and the mitophagy-related proteins Parkin and PINK1 in HC (#1-3) and DM platelets (#1-8).B Quantification of autophagy markers and mitophagy-related proteins from HC (#1-5) and DM (#1-18) individuals. (Beclin1; **p=0.0003, ATG12; *p=0.0388, ATG3; *p=0.0213, ATG7; p=0.4051, LC3; *p=0.0298, PINK1; *p=0.0155, Parkin; *p=0.0103 vs. HC). GAPDH served as a loading control.


C three panels represent autophagy stages found in DM platelets . Autophagosome structures containing / enclosing organelles are indicated by black box . Shown for each panel is an enlargement of autophagosome ( DM1 2 ) or autolysosome ( DM3 ) . Dashed structures are an outline of autophagosome membranes .

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C The three panels represent autophagy stages found in DM platelets. Autophagosome structures containing/enclosing organelles are indicated by the black box. Shown for each panel is an enlargement of the autophagosome (DM1 and 2) or the autolysosome (DM3). Dashed structures are an outline of the autophagosome membranes.


D LC3 immuno - EM of HC DM platelets . Arrows indicate immunogold - labeled LC3 clusters . No clusters were found in HC platelets . Representative areas of clusters of gold labeling in DM ( DM1 - 3 ) are presented . Shown in insets are enlargements of gold clusters adjacent to mitochondria - like structures .

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D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures.


E Double for MitoTracker ( mitochondria potential ) ( 50nM for 15 min ) LC3 ( autophagy ) in HC DM platelets . Boxed platelets are enlarged in Panel F . F A typical HC platelet has high potential low LC3 in contrast to a representative DM platelet which has low potential high LC3 . G of autophagosome positive platelets using 6 HC 4 DM samples after using CoxIV ( mitochondria ) LC3 ( autophagy marker ) . y - axis indicates of LC3 positive in total ( * * p = 0 . 006 vs . HC ) .

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E Double staining for MitoTracker (mitochondria membrane potential) (50nM for 15 min) and LC3 (autophagy) in HC and DM platelets. Boxed platelets are enlarged in Panel F.F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3.G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV (mitochondria) and LC3 (autophagy marker). The y-axis indicates percentage of LC3 positive cells in total cells (**p=0.006 vs. HC).


A Representative western blot of recognized autophagy signaling molecules ( pJNK , JNK , pAKT , AKT , pmTOR , mTOR ) in HC ( # 1 - 3 ) DM ( # 1 - 8 ) platelets . of autophagy upstream signaling molecules on HC ( # 1 - 5 ) DM ( # 1 - 18 ) individuals . ( pAKT / AKT ; p = 0 . 0747 , pJNK / JNK ; * p = 0 . 001 , pmTOR / mTOR ; p = 0 . 3356 vs . HC ) . GAPDH was used as loading control .

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A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control.


B Representative western blot using HC HG platelets with or without H2O2 . assessing for LC3I / II . GAPDH was used as loading control .

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B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control.


C Platelet suspensions were incubated in HG for 2 hours with H2O2 ( 1mM ) . ROS were evaluated using a detection kit ( Enzo Life Science ) for 60 mins at 37 ºC .

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C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC.


D Western blot of p53 , JNK LC3I / II in HC platelets treated with H2O2 ( 1mM for 1 hour ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as loading control .

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D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control.


E Confocal microscopy was used to corroborate Western using triple for Mitotracker , CoxIV , LC3 in HC or H2O2 . insets highlight association of low potential with high of LC3 increased colocalization of Cox IV with LC3 in H2O2 - treated platelets .

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E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets.


F HC platelets were pretreated with H2O2 to induce autophagy then treated with or without SP600125 ( JNK inhibitor ) at 3 concentrations ( 1 , 5 10 µM ) to assess for inhibition of autophagy . This Western blot is representative of three independent experiments . of three independent experiments is provided at right . ( pJNK / JNK in H2O2 / SP600125 ; * * p = 0 . 0286 vs . H2O2 group , LC3II in H2O2 ; * p = 0 . 0241 vs . HC group , LC3II in H2O2 / SP600125 ; * * p = 0 . 0044 vs . H2O2 group , n = 3 for each group ) .

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F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group).


G Triple of CoxIV , LC3 , LAMP1 in HC H2O2 with / without SP600125 - treated platelets using confocal microscopy . Arrow indicates localization of LC3 LAMP1 . Graph indicates colocalization between LC3 , LAMP1 CoxIV signal . y - axis indicates fold change of colocalization between LC3 LAMP1 in mitochondria . Signal of each group was converted to fold compared with HC values ( H2O2 ; * p = 0 . 023 vs . HC , H2O2 / SP600125 ; * p = 0 . 04 vs . H2O2 group ) .

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G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).


A Western blot of LC3 apoptosis markers phosphorylated p53 , p53 , LAMP1 in HC platelets . Platelets were treated with high glucose ( 25mM ) , with or without CCCP ( 10µM ) . This Western blot is representative of three independent experiments . B of phosphorylated p53 LAMP1 ( HG ; * p = 0 . 018 vs DMSO group , HG / CCCP ; * * p = 0 . 001 vs HG group , n = 4 for each group ) .

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A Western blot analysis of LC3 and the apoptosis markers phosphorylated p53, p53, and LAMP1 in HC platelets. Platelets were treated with high glucose (25mM), with or without CCCP (10µM). This Western blot is representative of three independent experiments.B Quantification analysis of phosphorylated p53 and LAMP1 (HG; *p=0.018 vs DMSO group, HG/CCCP; **p=0.001 vs HG group, n=4 for each group).


C recognized autophagy inhibitor ( 2mM 3 - Methyadenine ; 3MA for 1h ) was used to treat DM platelets compared to HC assessing for phosphorylated p53 , p53 , LC3I / IID of phosphorylated p53 / p53 was performed . Each value indicated average band density from a total of three independent samples ( DM / 3MA ; * p = 0 . 026 vs . DM group ) .

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C The recognized autophagy inhibitor (2mM 3-Methyadenine; 3MA for 1h) was used to treat DM platelets compared to HC assessing for phosphorylated p53, p53, and LC3I/IID Quantification analysis of phosphorylated p53/p53 was performed. Each value indicated the average band density from a total of three independent samples (DM/3MA; *p=0.026 vs. DM group).


E Western blot of PLPs transfected with control or ATG3 siRNA , with or without treatment of 1 mM H2O2 . Each lane displayed a different transfected group . F of ATG3 , phosphorylated p53 ( ser15 ) , p53 , GAPDH in each group . ( ATG3 in ATG3 siRNA ; * * p = 0 . 0003 vs control group , pp53 in ATG3 siRNA / H2O2 ; * p = 0 . 0145 vs . H2O2 group ) .

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O

E Western blot analysis of PLPs transfected with control or ATG3 siRNA, with or without treatment of 1 mM H2O2. Each lane displayed a different transfected group.F Quantification analysis of ATG3, phosphorylated p53 (ser15), p53, and GAPDH in each group. (ATG3 in ATG3 siRNA; **p=0.0003 vs control group, pp53 in ATG3 siRNA/ H2O2; *p=0.0145 vs. H2O2 group).


A Western blot of phosphorylated p53 ( ser15 ) , p53 , LC3I / II , GAPDH in WT mouse platelets . Mouse platelets were treated high glucose ( 25mM ) with or without CCCP ( 10µM ) . B of phosphorylated p53 ( ser15 ) LC3 II ( pp53 in HG ; * * p = 0 . 009 vs WT group , pp53 in HG / CCCP ; * p = 0 . 022 vs HG group , LC3 II in HG / CCCP ; * p = 0 . 028 vs HG group , n = 3 for each group ) .

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A Western blot analysis of phosphorylated p53 (ser15), p53, LC3I/II, and GAPDH in WT mouse platelets. Mouse platelets were treated high glucose (25mM) with or without CCCP (10µM).B Quantification analysis of phosphorylated p53 (ser15) and LC3 II (pp53 in HG; **p=0.009 vs WT group, pp53 in HG/CCCP ; *p=0.022 vs HG group, LC3 II in HG/CCCP ; *p=0.028 vs HG group, n=3 for each group).


C ΔΨm platelet apoptosis were measured by flow cytometry in WT or WT _ H2O2 or WT _ H2O2 / 3MA in mouse platelets . ΔΨm was detected using TMRM apoptosis were assessed with Annexin - V ( PS externalization ) . Representative of n = 3 . D Graph indicates of TMRM positive in total population ( H2O2 ; * * p = 0 . 002 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 008 vs . not treated group , NS means no significance , n = 3 ) . E Graph indicates of Annexin V positive in total population ( H2O2 ; NS , p = 0 . 058 vs . not treated group , H2O2 / 3MA ; * * p = 0 . 004 vs . not treated group , * * p = 0 . 008 vs . H2O2 group , NS means no significance , n = 3 ) .

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C ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 or WT_ H2O2/3MA in mouse platelets. ΔΨm was detected using TMRM and apoptosis levels were assessed with Annexin-V (PS externalization). Representative of n=3.D Graph indicates the percentage of TMRM positive cells in total cell population (H2O2; **p=0.002 vs. not treated group, H2O2/3MA; **p=0.008 vs. not treated group, NS means no significance, n=3).E Graph indicates the percentage of Annexin V positive cell in total cell population (H2O2; NS, p=0.058 vs. not treated group, H2O2/3MA; **p=0.004 vs. not treated group, **p=0.008 vs. H2O2 group, NS means no significance, n=3).


F Representative western blot of LC3 I / II in in WT or WT / H2O2 or WT / H2O2 / 3MA in mouse platelets ( n = 3 ) .

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F Representative western blot analysis of LC3 I/II in analysis in WT or WT/H2O2 or WT/H2O2/3MA in mouse platelets (n=3).


G Representative western blot of phosphorylated p53 , Parkin in Parkin - / - mice compared to WT . of phosphorylated p53 Parkin ( Parkin ; * * p = 0 . 0001 vs . WT , pp53 ; * p = 0 . 019 vs . WT , n = 4 ) .

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G Representative western blot analysis of phosphorylated p53, and Parkin in Parkin -/- mice compared to WT. Quantification analysis of phosphorylated p53 and Parkin (Parkin; **p=0.0001 vs. WT, pp53; *p=0.019 vs. WT, n=4).


H ΔΨm platelet apoptosis were measured by flow cytometry in WT or WT _ H2O2 Parkin - / - or Parkin - / - _ H2O2 mouse platelets . ΔΨm was detected using TMRM ( WT _ H2O2 ; * * p = 0 . 002 vs . WT group , Parkin - / - _ H2O2 ; * * p = 1 . 61525E - 06 vs . Parkin - / - group , n = 3 ) I Apoptosis level was assessed with Annexin - V ( PS externalization ) . Graph indicates of Annexin V positive in total population ( Parkin - / - : * p = 0 . 010 vs . WT group , Parkin - / - _ H2O2 ; * * p = 0 . 008 vs . WT _ H2O2 ; group , Parkin - / - _ H2O2 ; * * p = 0 . 001 vs . Parkin - / - group , NS means no significance n = 3 )

O O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE B-ORGANISM B-CELL_TYPE O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

H ΔΨm and platelet apoptosis were measured by flow cytometry analysis in WT or WT_H2O2 and Parkin -/- or Parkin -/-_H2O2 mouse platelets. ΔΨm was detected using TMRM (WT_ H2O2; **p= 0.002 vs. WT group, Parkin-/-_ H2O2; **p= 1.61525E-06 vs. Parkin-/- group, n=3)I Apoptosis level was assessed with Annexin-V (PS externalization). Graph indicates the percentage of Annexin V positive cell in total cell population (Parkin-/- : *p=0.010 vs. WT group, Parkin-/-_ H2O2 ;**p= 0.008 vs. WT_ H2O2; group, Parkin-/-_ H2O2; **p=0.001 vs. Parkin-/- group, NS means no significance n=3)


J Western blot in WT Parkin - / - demonstrating inability to induce LC3 despite addition of H2O2 . Shown graphically are LC3II to LC3I ratios ( WT _ H2O2 ; * p = 0 . 028 vs . WT group , n = 3 )

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J Western blot analysis in WT and Parkin -/- demonstrating the inability to induce LC3 despite the addition of H2O2. Shown graphically are the LC3II to LC3I ratios (WT_ H2O2;*p=0.028 vs. WT group, n=3)


A Representative western blot of pp53 in WT ( n = 6 ) , DM ( n = 6 ) PINK1 - / - DM mice ( n = 3 ) . GAPDH served as loading control ( DM ; * * p = 0 . 006 vs . WT group , PINK1 - / - DM ; * * p = 0 . 004 vs . DM group ) .

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A Representative western blot analysis of pp53 in WT (n=6), DM (n=6) and PINK1-/-DM mice (n=3). GAPDH served as the loading control (DM ; **p=0.006 vs. WT group, PINK1-/-DM; **p=0.004 vs. DM group).


B Measurement of p - selectin translocation to by flow cytometry in WT ( n = 3 ) , DM ( n = 3 ) PINK1 - / - DM ( n = 3 ) miceplatelets . Graphs indicate mean ( MFI ) in each group . ( * p < 0 . 05 vs . WT or DM ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O 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 Measurement of p-selectin translocation to the cell membrane by flow cytometry analysis in WT (n=3), DM (n=3) and PINK1 -/- DM (n=3) miceplatelets. Graphs indicate mean fluorescence intensity (MFI) in each group. (*p<0.05 vs. WT or DM).


C of vessel occlusion time after carotid arterial flow measurement ( WT DM ; PINK1 - / - : * * p < 0 . 01 vs . WT or DM ) .

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C Quantification of vessel occlusion time after carotid arterial flow measurement (WT and DM; and PINK1 -/- : **p<0.01 vs. WT or DM).


D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury , indicative of occlusive thrombosis . Flow was plotted as of baseline flow before injury ( DM : n = 3 , PINK1 - / - DM ; n = 3 ) .

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D Plot of carotid arterial flow by Doppler flow probe after 2 min FeCl3 injury, indicative of occlusive thrombosis. Flow was plotted as percentage of baseline flow before injury (DM: n=3, PINK1-/- DM; n=3).


A BAK - 6H permeabilising activity is exacerbated by cBID . BAK - 6H ( mBAK ∆ C21 - 6H ) was loaded at indicated concentrations onto Ni - NTA liposomes ( 5 μM ) prior to addition of cBID . release of carboxyfluorescein was measured over time . Results representative of at least 3 independent experiments .

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A BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 μM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least 3 independent experiments.


B BAK - 6H oligomerises on liposomes induced by cBID . Liposomes were incubated with BAK - 6H ( 150 nM ) indicated of cBID or a BID BH3 peptide for 60 min . Liposomes were solubilised in digitonin BAK oligomers were analysed by BN - PAGE . In parallel , samples were tested at endpoint for liposome permeabilisation on release of flurorescent dextran , normalised to total of detergent permeabilised liposomes . Results representative of 3 independent experiments .

O B-GENEPROD O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O

B BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of flurorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of 3 independent experiments.


C BAK - 6H oligomerisation on liposomes is blocked by BCL - XL . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( WT , 90 nM ) or cBID M97A ( 90 nM ) that has reduced affinity for BCL - XL , in presence or absence of BCL - XL for 60 mins prior to of BAK oligomerisation by BN - PAGE . Results representative of 2 independent experiments . Liposome permeabilisation was monitored with indicated combinations of cBID ( 90 nM ) , BAK - 6H ( 150 nM ) BCL - XL ( 1 μM ) . Data is presented as mean of 2 independent experiments with release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK cBid was approximately 50 % of detergent - lysis maximum .

O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O

C BAK-6H oligomerisation on liposomes is blocked by BCL-XL. Liposomes were incubated with BAK-6H (150 nM) together with cBID (WT, 90 nM) or cBID M97A (90 nM) that has reduced affinity for BCL-XL, in the presence or absence of BCL-XL for 60 mins prior to analysis of BAK oligomerisation by BN-PAGE. Results representative of 2 independent experiments. Liposome permeabilisation was monitored with the indicated combinations of cBID (90 nM), BAK-6H (150 nM) and BCL-XL (1 μM). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


D BAK - 6H oligomerisation on liposomes involves its BH3 domain . Liposomes were incubated with BAK - 6H ( 150 nM ) together with cBID ( 90 nM ) . Antibody recognising BAK BH3 domain ( 4B5 ) or α1 - α2 loop ( 7D10 ) , as shown on structure of BAK ( PDB : 2IMS ) , were added ( 2 . 4 μg / μl ) either prior to ( pre ) or after ( post ) incubation with cBID for 60 mins . BAK : Ab is indicated ( arrowhead ) . 7D10 which recognises activated form of mBAK ( Iyer et al . , 2016 ) , bound to gel - shifted BAK when added either before or after activation with cBID . In contrast , 4B5 failed to appreciably gel - shift inactive BAK ( lanes 3 5 ) or BAK that was already activated ( lane 6 ) , but gel - shifted BAK when present during cBID activation ( lane 4 ) . Liposome permeabilisation was assessed after 20 min incubation with cBID ( 90 nM ) , BAK - 6H ( 150 nM ) antibodies 4B5 ( 2 . 5 μg ) or 7D10 ( 2 . 5 μg ) . Data is presented as mean of 2 independent experiments with release relative to detergent - lysed liposomes at a timepoint when permeabilisation with BAK cBid was approximately 50 % of detergent - lysis maximum .

O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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-SUBCELLULAR O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O

D BAK-6H oligomerisation on liposomes involves its BH3 domain. Liposomes were incubated with BAK-6H (150 nM) together with cBID (90 nM). Antibody recognising the BAK BH3 domain (4B5) or α1-α2 loop (7D10), as shown on the structure of BAK (PDB:2IMS), were added (2.4 μg/μl) either prior to (pre) or after (post) incubation with cBID for 60 mins. BAK:Ab complex is indicated (arrowhead). 7D10 which recognises the activated form of mBAK (Iyer et al., 2016), bound to and gel-shifted BAK when added either before or after activation with cBID. In contrast, 4B5 failed to appreciably gel-shift inactive BAK (lanes 3 and 5) or BAK that was already activated (lane 6), but gel-shifted BAK when present during cBID activation (lane 4). Liposome permeabilisation was assessed after 20 min incubation with cBID (90 nM), BAK-6H (150 nM) and antibodies 4B5 (2.5 μg) or 7D10 (2.5 μg). Data is presented as mean of 2 independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum.


B BAK , or BAK with cysteine introduced at indicated positions ( on a BAK ∆ Cys background ) were stably expressed in Bax - / - Bak - / - MEFs assess for BAK expression by immunoblotting ( inset panel ; * indicates non - specific band . ) apoptotic activity in response to etoposide treatment .

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B BAK, or BAK with cysteine introduced at the indicated positions (on a BAK∆Cys background) were stably expressed in Bax-/-Bak-/- MEFs and assess for BAK expression by immunoblotting (inset panel; * indicates non-specific band.) and apoptotic activity in response to etoposide treatment.


C E24 R169 are proximal in inactive BAK on mitochondria . Mitochondria - enriched fractions from were incubated with oxidant ( CuPhe ) induced intramolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

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C E24 and R169 are proximal in inactive BAK on mitochondria. Mitochondria-enriched membrane fractions from cells were incubated with oxidant (CuPhe) and induced intramolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


D E24 R169 dissociate during BAK activation . Mitochondria - enriched fractions from were incubated with cBID ( 100 nM ) prior to oxidant ( CuPhe ) induced intramolecular intermolecular disulphide linkage of BAK was assessed on non - reducing SDS - PAGE .

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D E24 and R169 dissociate during BAK activation. Mitochondria-enriched membrane fractions from cells were incubated with cBID (100 nM) prior to oxidant (CuPhe) and the induced intramolecular and intermolecular disulphide linkage of BAK was assessed on non-reducing SDS-PAGE.


C Bax - / - Bak - / - MEFs expressing indicated BAK variants were treated with doxycycline ( Dox , 3 h ) , incubated for a further 24 h in presence or absence of BH3 - mimetic compounds , was assessed by PI uptake .

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C Bax-/-Bak-/- MEFs expressing the indicated BAK variants were treated with doxycycline (Dox, 3 h), incubated for a further 24 h in the presence or absence of BH3-mimetic compounds, and cell death was assessed by PI uptake.


D Mutation of BH4 promotes BAK activating conformation change . were treated with doxycycline ( 3 h ) to induce BAK expression followed by incubation with BH3 - mimetics where indicated ( for 2 h ) conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK ( G3172 ) .

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D Mutation of BH4 promotes BAK activating conformation change. Cells were treated with doxycycline (3 h) to induce BAK expression followed by incubation with BH3-mimetics where indicated (for 2 h) and conformation change of BAK was assessed by intracellular flow cytometry with an antibody that recognises activated BAK (G3172).


D BAK wild - type , or BAK with an engineered α1 - α2 loop thrombin cleavage site were stably expressed in Bax - / - Bak - / - MEFs . fractions were incubated with thrombin prior to incubation with copper phenanthroline ( CuPhe ) to induce disulphide - linkage . Samples were run on SDS - PAGE under non - reducing or reducing conditions immunoblotted for BAK with an antibody recognising BAK N - terminus upstream of thrombin cleavage site ( aa23 - 38 ) or an antibody recognising BH3 domain ( 4B5 ) downstream of cleavage site . Schematic indicates nature of BAK detected by each antibody . Disulphide - linkage indicated by a hashed . Note that N - terminal portion of thrombin BAK could not be detected with N - terminal antibody unless it was intramolecularly disulphide - linked . * cBID ( 100 nM ) was added to activate BAK during thrombin cleavage . Data representative of two independent experiments .

O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-GENEPROD O O O 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-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O

D BAK wild-type, or BAK with an engineered α1-α2 loop thrombin cleavage site were stably expressed in Bax-/-Bak-/- MEFs. Membrane fractions were incubated with thrombin prior to incubation with copper phenanthroline (CuPhe) to induce disulphide-linkage. Samples were run on SDS-PAGE under non-reducing or reducing conditions and immunoblotted for BAK with an antibody recognising the BAK N-terminus upstream of the thrombin cleavage site (aa23-38) or an antibody recognising the BH3 domain (4B5) downstream of the cleavage site. Schematic indicates the nature of the BAK protein detected by each antibody. Disulphide-linkage indicated by a hashed line. Note that the N-terminal portion of thrombin cleaved BAK could not be detected with the N-terminal antibody unless it was intramolecularly disulphide-linked. *cBID (100 nM) was added to activate BAK during thrombin cleavage. Data representative of two independent experiments.


E Cleavage of BAK α1 - α2 loop potentiates MOMP . fractions from Bax - / - Bak - / - MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in presence or absence of cBID ( 10 nM ) for indicated times . ( P ) supernatant ( S ) fractions were separated immunoblotted for cytochrome c or BAK 4B5 .

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E Cleavage of the BAK α1-α2 loop potentiates MOMP. Membrane fractions from Bax-/-Bak-/- MEFs expressing BAK or BAKthrombin were incubated or not with thrombin in the presence or absence of cBID (10 nM) for the indicated times. Membrane (P) and supernatant (S) fractions were separated and immunoblotted for cytochrome c or BAK 4B5.


( c ) Representative of a clustering with indicated on left . GFP mCherry are consistently displayed in cyan magenta ,

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(c) Representative micrographs of a clustering assay with the proteins indicated on the left. GFP and mCherry fluorescence are consistently displayed in cyan and magenta, respectively


( a ) Top : schematic representation of STG - p62 construct . Bottom : characterization of several STG - p62 clones in comparison to parental Hap1

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(a) Top: schematic representation of the STG-p62 construct. Bottom: characterization of several STG-p62 clones in comparison to parental Hap1 cells


( b ) Wild type or STG - p62 Hap1 were mock or puromycin treated for 2 hours stained for ubiquitin . endogenous p62 was stained with an anti - p62 antibody in parental Hap1 , while of GFP tag was recorded in STG - p62 . Arrowheads indicate co - localizing

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(b) Wild type or STG-p62 Hap1 cells were mock or puromycin treated for 2 hours and stained for ubiquitin. The endogenous p62 was stained with an anti-p62 antibody in the parental Hap1 cells, while the fluorescence of the GFP tag was recorded in STG-p62 cells. Arrowheads indicate co-localizing puncta


( c ) Purified STG - p62 was tested for cluster formation in presence of 20µM GST - 4xUb . Averages SDs of three independent replicates are shown for + GST - 4xUb sample of two for STG - p62

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(c) Purified STG-p62 was tested for cluster formation in presence of 20µM GST-4xUb. Averages and SDs of three independent replicates are shown for the +GST-4xUb sample and of two for STG-p62 alone


( d , e ) FCS of diffusion ( d ) brightness ( e ) of GFP1 - 5 ruler of STG - p62 in cytoplasm of Hap1 . Dashed lines represent linear regression of GFP1 - 5 ruler data . Regression coefficients are indicated . For data points averages SDs ( on both axes for STG - p62 ) are shown ( see methods for a list of number of measurements performed for each construct ) . MW : molecular . D : diffusion coefficient ( µm2 / s )

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O 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-EXP_ASSAY O O O O O O

(d,e) FCS analysis of diffusion (d) and brightness (e) of the GFP1-5 ruler and of STG-p62 in the cytoplasm of Hap1 cells. Dashed lines represent the linear regression of the GFP1-5 ruler data. Regression coefficients are indicated. For all data points averages and SDs (on both axes for STG-p62) are shown (see methods for a list of the number of measurements performed for each construct). MW: molecular weight. D: diffusion coefficient (µm2/s)


( a ) Representative of cluster formation assays conducted with indicated

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(a) Representative micrographs of cluster formation assays conducted with the indicated proteins


( c ) of cluster formation assays conducted with indicated . mCherry - p62 was pre - incubated with indicated 4xUb variants , followed by addition of streptavidin immediately before

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(c) Quantification of cluster formation assays conducted with the indicated proteins. mCherry-p62 was pre-incubated with the indicated 4xUb variants, followed by the addition of streptavidin immediately before imaging


( d ) Representative of indicated samples taken 60 minutes after addition of

(d) Representative micrographs of the indicated samples taken 60 minutes after addition of streptavidin


( e ) of cluster formation assays conducted with indicated

(e) Quantification of cluster formation assays conducted with the indicated proteins


( f ) of cluster formation assays conducted with 2µM mCherry - p62 incubated with indicated of GST - GFP - 4xUb . Box : highest particle number per field detected in each sample was plotted against relative GST - GFP - 4xUb . Data points were fitted to a sigmoidal curve . regression coefficient is

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(f) Quantification of cluster formation assays conducted with 2µM mCherry-p62 incubated with the indicated concentration of GST-GFP-4xUb. Box: the highest particle number per field detected in each sample was plotted against the relative GST-GFP-4xUb concentration. Data points were fitted to a sigmoidal curve. The regression coefficient is indicated


( g ) of cluster formation assays conducted with GFP - p62 indicated GST - tagged ubiquitin

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(g) Quantification of cluster formation assays conducted with GFP-p62 and the indicated GST-tagged ubiquitin chains


( h - i ) of cluster formation assays performed with mCherry - p62 GST - 4xUb in presence of indicated free ubiquitin chains . Ubiquitin chains were either pre - mixed with p62 before addition of GST - 4xUb ( h ) , or added 3 minutes after addition of GST - 4xUb to p62 ( i )

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(h-i) Quantification of cluster formation assays performed with mCherry-p62 and GST-4xUb in presence of the indicated free ubiquitin chains. Ubiquitin chains were either pre-mixed with p62 before the addition of GST-4xUb (h), or added 3 minutes after the addition of GST-4xUb to p62 (i)


( j ) of cluster formation assays conducted with mCherry - 2xFKBP - p62 in presence of AP20187 , GST - 4xUB indicated Ub

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(j) Quantification of cluster formation assays conducted with mCherry-2xFKBP-p62 in presence of AP20187, GST-4xUB and the indicated Ub chains


( k ) Representative of glutathione beads coated with indicated GST - tagged incubated with indicated mCherry - p62 variants . In pictures brightness was adjusted to highlight profile of beads . For graphs averages SDs from at least three independent replicates are

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(k) Representative micrographs of glutathione beads coated with the indicated GST-tagged proteins and incubated with the indicated mCherry-p62 variants. In all pictures the brightness was adjusted to highlight the profile of the beads. For all graphs averages and SDs from at least three independent replicates are shown


( a ) mCherry - p62 / GST - GFP - 4xUb clusters were photobleached recovery in bleached was monitored . mCherry - tagged low complexity domain ( LCD ) of hnRNPA1 ( hnRNPA1LCD ) was included as a reference for a liquid droplet - forming . Left : representative of bleached particles . arrows indicate position orientation of kymographs shown in Figure EV4a . Right : of recovery . traces of particles analyzed from three independent replicates are displayed

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(a) mCherry-p62/GST-GFP-4xUb clusters were photobleached and fluorescence recovery in the bleached area was monitored. The mCherry-tagged low complexity domain (LCD) of hnRNPA1 (hnRNPA1LCD) was included as a reference for a liquid droplet-forming protein. Left: representative micrographs of bleached particles. White arrows indicate the position and orientation of the kymographs shown in Figure EV4a. Right: quantification of fluorescence recovery. Individual traces of all particles analyzed from three independent replicates are displayed


( b ) p62 in STG - p62 were photobleached recovery was monitored . Left : representative of bleached . squares indicate bleached . Insert : magnification of bleached . Right : of recovery . Averages SDs of 28 ( unbleached ) 29 ( bleached ) from three independent experiments are

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(b) p62 puncta in STG-p62 cells were photobleached and the fluorescence recovery was monitored. Left: representative micrographs of bleached cells. White squares indicate the bleached puncta. Insert: magnification of the bleached puncta. Right: quantification of fluorescence recovery. Averages and SDs of 28 (unbleached) and 29 (bleached) puncta from three independent experiments are shown


( c ) Representative electron of negatively stained p62 filaments incubated in presence or absence of GST - 4xUb for indicated

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(c) Representative electron micrographs of negatively stained p62 filaments incubated in presence or absence of GST-4xUb for the indicated times


( a ) Top : schematic representation of domain architecture of GFP - NBR1 construct . PB1 : Phox Bem1p domain , ZZ : Zinc finger , CC : coiled - coil , LIR : LC3 - interacting region , UBA : ubiquitin - associated domain . Bottom : GFP - NBR1 was recruited to glutathione beads coated with indicated GST fusion imaged by spinning disc microscopy . Representative of at least 70 beads imaged per sample from two independent experiments

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(a) Top: schematic representation of the domain architecture of the GFP-NBR1 construct. PB1: Phox and Bem1p domain, ZZ: Zinc finger, CC: coiled-coil, LIR: LC3-interacting region, UBA: ubiquitin-associated domain. Bottom: GFP-NBR1 was recruited to glutathione beads coated with the indicated GST fusion proteins and imaged by spinning disc microscopy. Representative micrographs of at least 70 beads imaged per sample from two independent experiments


( b - c ) mCherry - p62 variants were recruited to GFP - or GFP - NBR1 - coated beads imaged as in ( a ) ( b ) Representative of at least 70 beads per sample from three independent

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(b-c) mCherry-p62 variants were recruited to GFP- or GFP-NBR1-coated beads and imaged as in (a) (b) Representative micrographs of at least 70 beads per sample from three independent replicates


( d - e ) 2µM mCherry - p62 / or GFP - NBR1 were incubated in presence of 5µM GST - 4xUb . Representative ( d ) of particle number for each channel ( e ) are shown . Averages SDs from at least three independent experiments are

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(d-e) 2µM mCherry-p62 and/or GFP-NBR1 were incubated in presence of 5µM GST-4xUb. Representative micrographs (d) and quantification of particle number for each channel (e) are shown. Averages and SDs from at least three independent experiments are shown


( a ) Cluster formation assays were conducted in presence or absence of 20µM mCherry - LC3B or mCherry , either pre - mixed with p62 or added 1 " after addition of GST - 4xUb . of particle number ( left ) ( right ) are shown

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(a) Cluster formation assays were conducted in presence or absence of 20µM mCherry-LC3B or mCherry, either pre-mixed with p62 or added 1" after the addition of GST-4xUb. Quantification of particle number (left) and size (right) are shown


( b ) Cluster formation assays were conduced with mCherry - p62 wt or LIR mutant in presence of GST - 4xUb . Left : representative of samples two minutes after addition of GST - 4xUb . Middle : of particle . dashed indicates time point of displayed on left . Right : input gel of samples after reaction . relative position of marker bands is indicated . For graphs , averages SDs from at least three independent replicates are

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) Cluster formation assays were conduced with mCherry-p62 wt or LIR mutant in presence of GST-4xUb. Left: representative images of the samples two minutes after the addition of GST-4xUb. Middle: quantification of particle count. The dashed line indicates the time point of images displayed on the left. Right: input gel of the samples after reaction. The relative position of marker bands is indicated. For all graphs, averages and SDs from at least three independent replicates are shown


( c ) Top left : Western blots of lysates of CRISPR / Cas9 genome - edited STG - p62 wild type LIR mutant . Clone Lir1B was used for experiments shown in Figure 6c . Bottom left : Representative pictures of STG - p62 WT or LIR mutant Hap1 treated with Puromycin for 90 minutes stained with anti - Ub conjugates antibody ( FK2 ) . Right : of p62 per ( top ) , of their co - localization upon puromycin treatment ( bottom ) . Averages SDs from three independent experiments are shown . In total , at least 1440 number of were counted per

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD 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-GENEPROD O O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O 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) Top left: Western blots of lysates of CRISPR/Cas9 genome-edited STG-p62 wild type and LIR mutant cells. Clone Lir1B was used for the experiments shown in Figure 6c. Bottom left: Representative pictures of STG-p62 WT or LIR mutant Hap1 cells treated with Puromycin for 90 minutes and stained with anti-Ub conjugates antibody (FK2). Right: quantification of cytoplasmic p62 puncta per cell (top), and of their co-localization upon puromycin treatment (bottom). Averages and SDs from three independent experiments are shown. In total, at least 1440 number of cells were counted per condition


( A ) Solubilized lysosomes cross ‐ linked with DSP were subjected to affinity chromatography with antibody against tail of lamp2a ( lanes 1 2 ) . Eluted were subjected to SDS - PAGE silver stained ( lane 1 ) , or immunoblotted for lamp2a ( lane 2 ) . of N ‐ terminus of 32 kDa band is shown . Part of solubilized lysosomes were directly subjected to non ‐ reducing SDS - PAGE ( 5 % ) immunoblotted for lamp2a ( lane 3 ) or PPCA ( lane 4 ) . Arrowheads indicate lamp2a multimeric complexes . 600 kDa ( * ) 200 kDa ( * * ) molecular complexes of lamp2a were excised subjected to reducing SDS - PAGE ( 12 % ) silver ( lanes 5 6 ) . Molecular ( kDa ) is indicated on left .

O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(A) Solubilized lysosomes cross‐linked with DSP were subjected to affinity chromatography with the antibody against the cytosolic tail of lamp2a (lanes 1 and 2). Eluted proteins were subjected to SDS-PAGE and silver stained (lane 1), or immunoblotted for lamp2a (lane 2). The sequence of the N‐terminus of the 32 kDa band is shown. Part of the solubilized lysosomes were directly subjected to non‐reducing SDS-PAGE (5%) and immunoblotted for lamp2a (lane 3) or PPCA (lane 4). Arrowheads indicate lamp2a multimeric complexes. The 600 kDa (*) and 200 kDa (**) molecular weight complexes of lamp2a were excised and subjected to reducing SDS-PAGE (12%) and silver staining (lanes 5 and 6). Molecular weight (kDa) is indicated on the left.


( B ) Immunoblot of PPCA in rat liver lysosomes , their membranes ( MB ) matrices ( MTX ) ( 100 μg of ) incubated alone ( None ) or with 5 mM MgCl2 . Arrowheads indicate precursor ( 54 kDa ) heavy chain ( 32 kDa ) of mature PPCA .

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(B) Immunoblot analysis of PPCA in rat liver lysosomes, their membranes (MB) and matrices (MTX) (100 μg of protein) incubated alone (None) or with 5 mM MgCl2. Arrowheads indicate the precursor (54 kDa) and heavy chain (32 kDa) of the mature PPCA.


( C ) Immunoblot of hsc73 ( upper ) PPCA ( lower ) in membranes from rat liver lysosomes incubated alone ( None ) or with 5 mM MgCl2 , CaCl2 , MnCl2 or 2 mM EDTA .

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(C) Immunoblot analysis of hsc73 (upper) and PPCA (lower) in membranes from rat liver lysosomes incubated alone (None) or with 5 mM MgCl2, CaCl2, MnCl2 or 2 mM EDTA.


( D ) Immunoblot of lamp2a co ‐ immunoprecipitated with PPCA in absence of chemical cross ‐ linkers from membranes of liver lysosomes incubated as in ( B ) . heavy chains of IgG are shown . Lane 1 contains 1 / 10 of total amount of lysosomal membranes used for immunoprecipitation .

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(D) Immunoblot analysis of lamp2a co‐immunoprecipitated with PPCA in the absence of chemical cross‐linkers from membranes of liver lysosomes incubated as in (B). Light and heavy chains of IgG are shown. Lane 1 contains 1/10 of the total amount of lysosomal membranes used for immunoprecipitation.


( E ) Membranes from lysosomes treated as in ( D ) were subjected to immunoprecipitation with an antibody specific for lamp2a or with a pre ‐ immune serum . Carboxypeptidase activity in total lysosomes in immunoprecipitates was measured as described under Materials methods . Values are expressed as of total carboxypeptidase activity in lysosomes present in immunoprecipitates are mean ± SE of three different experiments .

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(E) Membranes from lysosomes treated as in (D) were subjected to immunoprecipitation with an antibody specific for lamp2a or with a pre‐immune serum. Carboxypeptidase activity in total lysosomes and in the immunoprecipitates was measured as described under Materials and methods. Values are expressed as percentage of total carboxypeptidase activity in lysosomes present in the immunoprecipitates and are the mean ± SE of three different experiments.


of PPCA associated with lysosomal correlate with rates of CMA . Membranes of lysosomes incubated with increasing concentrations of GAPDH ( A ) or MgCl2 ( B , top panel ) as labeled , were analyzed by immunoblot for forms of lamp2 ( upper ) , lamp2a ( middle ) or PPCA ( lower ) . arrowhead indicates a previously identified truncated form of lamp2 lacking / transmembrane region ( Cuervo Dice , 2000b ) . ( B , bottom panel ) Chart showing effect of increasing concentrations of MgCl2 , CaCl2 or EDTA on degradation of [ 14C ] GAPDH by intact ratliver lysosomes . Values are means ± SE of six different experiments . Differences from control value were significant at P 0 . 001 ( * * ) or P 0 . 01 ( * ) .

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Levels of PPCA associated with the lysosomal membrane correlate with rates of CMA. Membranes of lysosomes incubated with increasing concentrations of GAPDH (A) or MgCl2 (B, top panel) as labeled, were analyzed by immunoblot for all forms of lamp2 (upper), lamp2a (middle) or PPCA (lower). The arrowhead indicates a previously identified truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). (B, bottom panel) Chart showing the effect of increasing concentrations of MgCl2, CaCl2 or EDTA on the degradation of [14C]GAPDH by intact ratliver lysosomes. Values are means ± SE of six different experiments. Differences from the control value were significant at P 0.001 (**) or P 0.01 (*).


( C ) Immunoblot for PPCA ( upper ) cathepsin L ( lower ) of rat liver lysosomes ( 100 μg of ) with high ( H + ) low ( H − ) activity for CMA of lysosomal membranes ( L . Memb ) matrices ( L . Mtx ) from both groups of lysosomes ( 200 μg of ) .

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(C) Immunoblot analysis for PPCA (upper) and cathepsin L (lower) of rat liver lysosomes (100 μg of protein) with high (H+) and low (H−) activity for CMA and of the lysosomal membranes (L.Memb) and matrices (L.Mtx) from both groups of lysosomes (200 μg of protein).


( D ) Immunoblot for lamp2a , PPCA cathepsin D , as labeled , of lysosomes ( 100 μg of ) from fed or 48 h starved rats membranes ( L . Memb ) matrices ( L . Mtx ) of both goups of lysosomes ( 200 μg of ) . Only mature forms of cathepsins are

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(D) Immunoblot analysis for lamp2a, PPCA and cathepsin D, as labeled, of lysosomes (100 μg of protein) from fed or 48 h starved rats and the membranes (L. Memb) and matrices (L. Mtx) of both goups of lysosomes (200 μg of protein). Only mature forms of the cathepsins are shown


( E ) Correlation between CMA activity , measured as in ( B , bottom panel ) of PPCA at lysosomal determined by denstometric of immunoblots similar to those shown in Figures 1 2 in lysosomes from fed or 20 h starved rats ( Starv 20h ) , highly active ( H + ) or less active ( H − ) lysosomes , or lysosomes incubated with 1 mM CaCl2 , MgCl2 , EDTA , or increasing amounts ( 2 , 5 , 10 μg ) of GAPDH . Values are expressed as times value in fed rats ( control ) are means ± SE of 3 - 6 different experiments .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

(E) Correlation between CMA activity, measured as in (B, bottom panel) and levels of PPCA at the lysosomal membrane determined by denstometric quantification of immunoblots similar to those shown in Figures 1 and 2 in lysosomes from fed or 20 h starved rats (Starv 20h), highly active (H+) or less active (H−) lysosomes, or lysosomes incubated with 1 mM CaCl2, MgCl2, EDTA, or increasing amounts (2, 5, 10 μg) of GAPDH. Values are expressed as times the value in fed rats (control) and are means ± SE of 3-6 different experiments.


( A ) Rates of degradation in skin fibroblasts from wild ‐ type ( control ) or PPCA ( − / − ) mice maintained in presence ( serum + ) or absence of serum ( serum − ) . Values are expressed as of initially radiolabeled remaining at each time are means ± SE of four different experiments .

O O O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O 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) Rates of protein degradation in skin fibroblasts from wild‐type (control) or PPCA(−/−) mice maintained in the presence (serum +) or absence of serum (serum −). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of four different experiments.


( B ) Degradation of [ 14C ] GAPDH after 30 min incubation with lysosomes isolated from wild ‐ type PPCA ( − / − ) mice skin fibroblasts supplemented or deprived of serum was measured as described in Material methods . Where indicated , 25 μg of RNase A were added . Values are means ± SE of four different experiments . Differences from control value were significant to P 0 . 001 ( * * ) .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-ORGANISM B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O 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) Degradation of [14C]GAPDH after 30 min incubation with lysosomes isolated from wild‐type and PPCA(−/−) mice skin fibroblasts supplemented or deprived of serum was measured as described in Material and methods. Where indicated, 25 μg of RNase A were added. Values are means ± SE of four different experiments. Differences from the control value were significant to P 0.001 (**).


( C ) Immunofluorescent for lamp2a lamp1 of skin fibroblasts from wild ‐ type PPCA ( − / − ) mice . Gain for lamp2a ( top ) was 1 / 8 of one for lamp1 ( bottom ) to visualize punctate pattern of lamp2a in PPCA ( − / − ) mice . Inset : detail of vesicular distribution of lamp2a . gain in wild type was eight times gain in PPCA ( − / − ) mice ) . Bars : 100 μm ( top ) , 50 μm ( bottom ) , 10 μm ( inset ) .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-TISSUE B-CELL_TYPE O O O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

(C) Immunofluorescent staining for lamp2a and lamp1 of skin fibroblasts from wild‐type and PPCA(−/−) mice. Gain for lamp2a images (top) was 1/8 of the one for lamp1 (bottom) to visualize the punctate pattern of lamp2a in PPCA(−/−) mice. Inset: detail of the vesicular distribution of lamp2a. The gain in wild type was eight times the gain in PPCA(−/−) mice). Bars: 100 μm (top), 50 μm (bottom), 10 μm (inset).


( D ) Immunoblot for lamp2a , lamp2s , lamp1 or cathepsin D ( cath D ) of homogenates ( 75 μg of ) , lysosomes ( 10 μg of ) lysosomal membranes ( L . MB ) matrices ( L . MTX ) ( 5 μg of ) isolated from wild ‐ type ( WT ) PPCA ( − / − ) skin fibroblasts . bottom chart shows densitometric of ( means + SE ) from four immunoblots similar to those shown here . Differences from control value were significant to P 0 . 001 ( * * ) P 0 . 05 ( * ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O 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) Immunoblot analysis for lamp2a, all lamp2s, lamp1 or cathepsin D (cath D) of homogenates (75 μg of protein), lysosomes (10 μg of protein) and lysosomal membranes (L.MB) and matrices (L.MTX) (5 μg of protein) isolated from wild‐type (WT) and PPCA(−/−) skin fibroblasts. The bottom chart shows the densitometric quantification of the membrane proteins (means + SE) from four immunoblots similar to those shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*).


( A ) Immunoblot for lamp2a , lamp2s , lamp1 , transferrin receptor ( TFR ) or cathepsin D ( cath D ) of liver homogenates ( 100 μg of ) from wild ‐ type ( WT ) , PPCA ( − / − ) β ‐ galactosidase ( − / − ) ( GM ) mice . Right : densitometric of three immunoblots ( means + SE ) similar to ones shown here . Differences from control value were significant to P 0 . 001 ( * * ) P 0 . 05 ( * ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O O O 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) Immunoblot analysis for lamp2a, all lamp2s, lamp1, transferrin receptor (TFR) or cathepsin D (cath D) of liver homogenates (100 μg of protein) from wild‐type (WT), PPCA(−/−) and β‐galactosidase(−/−) (GM) mice. Right: densitometric quantification of three immunoblots (means + SE) similar to the ones shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*).


( B ) Immunocytochemistry for lamp2a ( left ) , lamp1 ( center ) or lamp2s ( right ) in hematoxylin eosin ‐ stained sections from same livers . Bar : 50 μm . Black arrows : hepatocytes ; arrows : Kupffer .

O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O

(B) Immunocytochemistry for lamp2a (left), lamp1 (center) or lamp2s (right) in hematoxylin and eosin‐stained sections from the same livers. Bar: 50 μm. Black arrows: hepatocytes; white arrows: Kupffer cells.


( C ) Immunofluorescence for lamp2a ( top ) P ‐ glycoprotein ( bottom ) in mouse liver sections . right panel shows merged . Bar : 100 μm . Inset shows detailed at higher magnification . Bar : 50 μm .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-ORGANISM B-TISSUE 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) Immunofluorescence for lamp2a (top) and P‐glycoprotein (bottom) in mouse liver sections. The right panel shows merged images. Bar: 100 μm. Inset shows detailed area at higher magnification. Bar: 50 μm.


( A ) Autofluorography of sequential immunoprecipitation of lamp2a first ( left ) remaining isoforms of lamp2 second ( right ) from metabolically labeled wild ‐ type PPCA ( − / − ) mouse skin fibroblasts . Values are means ± SE of three different experiments similar to ones shown in upper panels .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O

(A) Autofluorography of the sequential immunoprecipitation of lamp2a first (left) and all the remaining isoforms of lamp2 second (right) from metabolically labeled wild‐type and PPCA(−/−) mouse skin fibroblasts. Values are means ± SE of three different experiments similar to the ones shown in the upper panels.


( B ) Immunoblot for lamp2a ( top ) lamp2 isoforms ( bottom ) in lysosomalmembranes from wild ‐ type ( WT ) or PPCA ( − / − ) mouseskinfibroblasts at different times after incubation at 37 ° C . open arrowhead indicates truncated form of lamp2 lacking / transmembrane region ( Cuervo Dice , 2000b ) . Right shows densitometric ( mean ± SE ) of four different experiments similar to one shown here . Values are expressed as of remaining lamp2a , are means ± 1 SE of three different experiments .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O

(B) Immunoblot analysis for lamp2a (top) and all lamp2 isoforms (bottom) in lysosomalmembranes from wild‐type (WT) or PPCA(−/−) mouseskinfibroblasts at different times after incubation at 37°C. The open arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). Right shows the densitometric quantification (mean ± SE) of four different experiments similar to the one shown here. Values are expressed as percentage of remaining lamp2a, and are means ± 1 SE of three different experiments.


( A ) Rates of total degradation in presence absence of serum of normal skin fibroblasts ( CTR ) , GS ( None ) GS supplemented with precursor forms of native PPCA ( + PPCA ) , a catalytic mutant of PPCA ( + PPCA S / A ) or cathepsin L ( Cath L ) . Values are means ± SE of six different experiments . Differences from control value were significant for P 0 . 001 ( * * ) P 0 . 05 ( * ) .

O O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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) Rates of total protein degradation in the presence and absence of serum of normal skin fibroblasts (CTR), GS cells (None) and GS cells supplemented with the precursor forms of native PPCA (+PPCA), a catalytic mutant of PPCA (+PPCA S/A) or cathepsin L (Cath L). Values are means ± SE of six different experiments. Differences from the control value were significant for P 0.001 (**) and P 0.05 (*).


( B ) Immunoblot for lamp2a at time 0 or after 40 min incubation at 37 ° C of lysosomes isolated from PPCA ( − / − ) ( None ) PPCA ( − / − ) supplemented as in ( A ) . At 40 min immunoblot with antibody against luminal region of lamp2a is depicted to show truncated form of lamp2a ( open arrowhead ) . Right : densitometric for lamp2a ( means + range of values ) of two different experiments similar to one shown . Values are expressed as of lamp2a present in non ‐ supplemented .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O

(B) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.


( C ) Immunoblot for lamp2a at time 0 or after 40 min incubation at 37 ° C of lysosomes isolated from PPCA ( − / − ) ( None ) PPCA ( − / − ) supplemented as in ( A ) . At 40 min immunoblot with antibody against luminal region of lamp2a is depicted to show truncated form of lamp2a ( open arrowhead ) . Right : densitometric for lamp2a ( means + range of values ) of two different experiments similar to one shown . Values are expressed as of lamp2a present in non ‐ supplemented .

(C) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.


( D ) Immunocytochemistry for lamp2a of liver ( top ) kidney ( bottom ) sections of ‐ matched wild ‐ type ( WT ) , PPCA ( − / − ) bone marrow transplanted ( BMT ) mice . Bar , 50 μm ( liver ) or 100 μm ( kidney ) .

O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O O O O B-TISSUE O O O O O B-TISSUE O O

(D) Immunocytochemistry for lamp2a of liver (top) and kidney (bottom) sections of age‐matched wild‐type (WT), PPCA(−/−) and bone marrow transplanted (BMT) mice. Bar, 50 μm (liver) or 100 μm (kidney).


( A ) Left : acid sequences of C ‐ terminal regions of lamp2a from different species of other lamp2 isoforms lamp1 from mouse . proposed cleavage site for PPCA in lamp2a is highlighted . Right : immunoblot for HA of lysosomal membranes isolated from two different clones of HEK293 ( mouse ; upper panel ) NIH 3T3 ( human ; lower panel ) stably transfected with wild ‐ type ( FL ) mutated ( GL ) HA ‐ lamp2a . arrowhead indicates truncated form of lamp2 lacking / transmembrane region .

O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-CELL_LINE O B-ORGANISM O O O O B-CELL_LINE I-CELL_LINE O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O

(A) Left: amino acid sequences of the C‐terminal regions of lamp2a from different species and of other lamp2 isoforms and lamp1 from mouse. The proposed cleavage site for PPCA in lamp2a is highlighted. Right: immunoblot analysis for HA of lysosomal membranes isolated from two different clones of HEK293 (mouse; upper panel) and NIH 3T3 cells (human; lower panel) stably transfected with wild‐type (FL) and mutated (GL) HA‐lamp2a. The arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region.


( B ) Total rates of degradation in presence absence of serum in non ‐ transfected HEK293 ( WT ) in transfected HEK293 clones described in ( A ) . Values are expressed as of initially radiolabeled remaining at each time are means ± SE of three different experiments .

O O O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O O O B-CELL_LINE O O O 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) Total rates of protein degradation in the presence and absence of serum in non‐transfected HEK293 cells (WT) and in the transfected HEK293 clones described in (A). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of three different experiments.


( C ) Top : immunoblot with an anti ‐ HA antibody of purified wild ‐ type ( FL ) mutated ( GL ) mouse HA ‐ lamp2a incubated in an acidic pH buffer alone , or with PPCA . Bottom : wild ‐ type HA ‐ lamp2a was incubated as in top panel without additions ( None ) or in presence of 2 mM EDTA , 10 μM L ‐ trans ‐ epoxysuccinyl ‐ leucylamide ‐ ( 4 ‐ guanido ) ‐ butane ( E ‐ 64 ) , 2 μM pepstatin A ( Pepst ) or 10 μM ABSF . open arrowhead indicates truncated form of lamp2a .

O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O

(C) Top: immunoblot analysis with an anti‐HA antibody of purified wild‐type (FL) and mutated (GL) mouse HA‐lamp2a incubated in an acidic pH buffer alone, or with PPCA. Bottom: wild‐type HA‐lamp2a was incubated as in the top panel without additions (None) or in the presence of 2 mM EDTA, 10 μM L‐trans‐epoxysuccinyl‐leucylamide‐(4‐guanido)‐butane (E‐64), 2 μM pepstatin A (Pepst) or 10 μM ABSF. The open arrowhead indicates the truncated form of lamp2a.


( D ) Electron microscopy from purified 40S 60S ribosomal complexes ( 1 pmol each ) assembled in vitro in presence of MgCl2 , in presence or absence of 5 pmol of ( PR ) 20 . Assembled 80S particles are indicated ( red arrows ) . Scale bar ( ) represents 10 nm . ( E ) of 80S particles identified in ( D ) ( n = 1000 ) in non - assembly ( 1 mM MgCl2 ) or assembly ( 5 mM MgCl2 ) conditions . (

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O

(D) Electron microscopy images from purified 40S and 60S ribosomal complexes (1 pmol each) assembled in vitro in the presence of MgCl2, and in the presence or absence of 5 pmol of (PR)20. Assembled 80S particles are indicated (red arrows). Scale bar (white) represents 10 nm. (E) Quantification of 80S particles identified in (D) (n=1000) in non-assembly (1 mM MgCl2) or assembly (5 mM MgCl2) conditions. (


( C , D ) High - throughput microscopy ( HTM ) - mediated of 5 - ethynyl - uridine ( EU ) ( C ) polyA + mRNA ( D ) per nucleus found in U2OS exposed to 10 μM ( PR ) 20 for 16 h . EU was added 30 ´ prior to fixation ( n = 3 ) . Black lines indicate median values in both panels . In D , boxes define upper lower quartiles whiskers mark highest lowest observations . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - . Representative from these analyses are provided in each case . Scale bar ( ) indicates 2 - 5 μm .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) High-throughput microscopy (HTM)-mediated analysis of 5-ethynyl-uridine (EU) (C) and polyA+ mRNA (D) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 16 h. EU was added 30´ prior to fixation (n=3). Black lines indicate median values in both panels. In D, boxes define upper and lower quartiles and whiskers mark the highest and lowest observations. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.


( E ) Effect of ( PR ) 20 ( 10 μM , 16 h ) in number of Cajal Bodies ( CB ) identified by immunofluorescence with anti - COILIN anti - SMN antibodies ( n = 3 ) . Data represent mean values ± SD . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - . Representative from these analyses are provided in each case . Scale bar ( ) indicates 2 - 5 μm .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of (PR)20 (10 μM, 16 h) in the number of Cajal Bodies (CB) identified by immunofluorescence with anti-COILIN and anti-SMN antibodies (n=3). Data represent mean values ± SD. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test. Representative images from these analyses are provided in each case. Scale bar (white) indicates 2-5 μm.


( B , C ) of GAPDH or 5 . 8 rRNA quantified by qPCR in reactions containing increasing doses of ( PR ) 20 ( n = 3 ) . Data represent mean values ± . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; t - .

O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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, C) Percentage of GAPDH or 5.8 rRNA levels quantified by qPCR in reactions containing increasing doses of (PR)20 (n=3).Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001; ****, p<0.0001; t-test.


( D ) HTM - mediated of 5 - ethynyl - deoxyuridine ( EdU ) per nucleus found in U2OS exposed to 10 μM ( PR ) 20 for 4 h . EdU was added 30 ´ prior to fixation . Representative from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( ) indicates 5 μm .

O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O 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) HTM-mediated analysis of 5-ethynyl-deoxyuridine (EdU) levels per nucleus found in U2OS cells exposed to 10 μM (PR)20 for 4 h. EdU was added 30´ prior to fixation. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( E ) HTM - mediated of γH2AX per nucleus in U2OS exposed to 3Gy of IR in presence or absence of 10 μM ( PR ) 20 for indicated times . Note accumulation of with persistent damage ( γH2AX ) 10 h after IR in presence of DPR . Representative from these analyses are provided ( right ) . Black lines indicate median values . Scale bar ( ) indicates 5 μm .

O O O B-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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) HTM-mediated analysis of γH2AX levels per nucleus in U2OS cells exposed to 3Gy of IR in the presence or absence of 10 μM (PR)20 for the indicated times. Note the accumulation of cells with persistent DNA damage (γH2AX) 10 h after IR in the presence of the DPR. Representative images from these analyses are provided (right). Black lines indicate median values. Scale bar (white) indicates 5 μm.


( D ) Immunofluorescence of Cy3 - labelled salmon protamine ( red ) nucleolar factor UBF1 ( green ) in U2OS treated with 30 μM Cy3 - protamine for 4 h . Scale bar ( ) indicates 2 . 5 μm .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-ORGANISM B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(D) Immunofluorescence of Cy3-labelled salmon protamine (red) and the nucleolar factor UBF1 (green) in U2OS cells treated with 30 μM Cy3-protamine for 4 h. Scale bar (white) indicates 2.5 μm.


( E , F ) HTM - mediated of EU per nucleus ( E ) O - propargyl - puromycin ( OPP ) per ( F ) in U2OS exposed to 30 μM protamine for 16 ( E ) or 24 ( F ) h . EU OPP were added 30 ´ prior to fixation . Black lines indicate median values ( * * * * , p < 0 . 0001 ; t - ) .

O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O 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) HTM-mediated quantification of the EU levels per nucleus (E) and O-propargyl-puromycin (OPP) levels per cell (F) in U2OS cells exposed to 30 μM protamine for 16 (E) or 24 (F) h. EU and OPP were added 30´ prior to fixation. Black lines indicate median values (****, p<0.0001; t-test).


( D ) Immunofluorescence of HA - ( PR ) 20 ( green ) nucleolar factor UBF1 ( red ) in U2OS treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h . Data information : Scale bar ( ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - .

O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O 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) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 2 μM of a 19 nt ssDNA oligonucleotide for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( E ) Immunofluorescence of HA - ( PR ) 20 ( green ) Cy5 ( red ) in U2OS treated with 7 . 5 μM HA - ( PR ) 20 alone or together with 4 μM of Cy5 - labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h . Data information : Scale bar ( ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - .

O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O 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) Immunofluorescence of HA-(PR)20 (green) and Cy5 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with 4 μM of Cy5-labeled 19 nt ssDNA or ssRNA oligonucleotides for 8 h. Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.


( F ) Clonogenic survival of U2OS exposed to 7 . 5 μM with indicated peptides or protamine ( PROT . ) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0 . 5 μM Heparin .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O

(F) Clonogenic survival assay of U2OS cells exposed to 7.5 μM with the indicated peptides or protamine (PROT.) alone or together with 2 μM of a 38 nt ssDNA oligonucleotide or 0.5 μM Heparin.


( G ) Immunofluorescence of HA - ( PR ) 20 ( green ) nucleolar factor UBF1 ( red ) in U2OS treated with 7 . 5 μM HA - ( PR ) 20 alone or together with heparin ( 0 . 5 μM ) . Data information : Scale bar ( ) indicates 2 . 5 μm . * , p < 0 . 1 ; * * * , p < 0 . 001 ; t - .

O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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) Immunofluorescence of HA-(PR)20 (green) and the nucleolar factor UBF1 (red) in U2OS cells treated with 7.5 μM HA-(PR)20 alone or together with heparin (0.5 μM). Data information: Scale bar (white) indicates 2.5 μm. *, p<0.1; ***, p<0.001; t-test.