Figure 4A 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 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 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 ) .

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 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 O O O O O O O

Figure 4A 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 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 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).


Figure 5A 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 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 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 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 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 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 )

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

Figure 5A 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 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 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 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 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 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)


Figure 6A 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 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 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 percentage of baseline flow before injury ( DM : n = 3 , PINK1 - / - DM ; n = 3 ) .

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Figure 6A 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 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 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 percentage of baseline flow before injury (DM: n=3, PINK1-/- DM; n=3).


Figure 1A 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 ) 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 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 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 .

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

Figure 1A 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) 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 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 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.


Figure 3B 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3B 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 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 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.


Figure 4C 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 . 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 ) .

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Figure 4C 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. 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).


Figure 5D 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 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 .

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

Figure 5D 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 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.


Figure 1 ( 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 ,

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Figure 1(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


Figure 2 ( 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 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 and SDs of three independent replicates are shown for the + GST - 4xUb sample and of two for STG - p62 alone ( 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 )

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

Figure 2(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 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 and SDs of three independent replicates are shown for the +GST-4xUb sample and of two for STG-p62 alone(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)


Figure 3 ( a ) Representative micrographs of cluster formation assays conducted with the indicated proteins ( 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 micrographs of the indicated samples taken 60 minutes after addition of streptavidin ( e ) Quantification of cluster formation assays conducted with the indicated proteins ( 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 ) Quantification of cluster formation assays conducted with GFP - p62 and the indicated GST - tagged ubiquitin chains ( 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 ) Quantification of cluster formation assays conducted with mCherry - 2xFKBP - p62 in presence of AP20187 , GST - 4xUB and the indicated Ub chains ( 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

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

Figure 3 (a) Representative micrographs of cluster formation assays conducted with the indicated proteins (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 micrographs of the indicated samples taken 60 minutes after addition of streptavidin(e) Quantification of cluster formation assays conducted with the indicated proteins(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) Quantification of cluster formation assays conducted with GFP-p62 and the indicated GST-tagged ubiquitin chains(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) Quantification of cluster formation assays conducted with mCherry-2xFKBP-p62 in presence of AP20187, GST-4xUB and the indicated Ub chains(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


Figure 4 ( 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 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 micrographs of negatively stained p62 filaments incubated in presence or absence of GST - 4xUb for the indicated

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

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


Figure 5 ( 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 and imaged as in ( a ) ( b ) Representative micrographs of at least 70 beads per sample from three independent replicates ( 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

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

Figure 5 (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 and imaged as in (a) (b) Representative micrographs of at least 70 beads per sample from three independent replicates(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


Figure 6 ( 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 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 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

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

Figure 6 (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 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 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


Figure 1 ( 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 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 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 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 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 .

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

Figure 1(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 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 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 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 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.


Figure 2Levels 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 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 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 ) 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 .

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 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 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

Figure 2Levels 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 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 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) 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.


Figure 3 ( 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 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 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 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 ( * ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O 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) 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 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 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 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 (*).


Figure 4 ( 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 and eosin ‐ stained sections from the same livers . Bar : 50 μm . Black arrows : hepatocytes ; white arrows : Kupffer cells . ( 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 .

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 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

Figure 4(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 and eosin‐stained sections from the same livers. Bar: 50 μm. Black arrows: hepatocytes; white arrows: Kupffer cells.(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.


Figure 5 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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.


Figure 6 ( 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 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 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 ) 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 ) .

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 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 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 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

Figure 6(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 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 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) 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).


Figure 7 ( 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 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 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 .

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 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 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

Figure 7(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 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 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.


Figure 1 ( 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 . (

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

Figure 1(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. (


Figure 2 ( 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 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 .

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

Figure 2(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 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.


Figure 3 ( 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 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 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 .

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 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 O O O O O O O O O O

Figure 3(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 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 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.


Figure 4 ( 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 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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).


Figure 6 ( 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 ) 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 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 ) 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 .

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Figure 6(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) 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 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) 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.


Figure 7 ( A ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without 2 μM of a non - coding 38nt oligonucleotide . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . Note that besides its impact on overall survival , the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide . ( B , C ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( B ) and NSC34DIFF ( C ) treated as in ( A ) ( n = 3 ) . Data represent mean values ± SEM . ( Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 . ( D ) Immunofluorescence of the neuronal marker TUBB3 ( red ) in mouse motor neurons differentiated from mESC ( mMN ) or from NSC34 cells ( NSC34DIFF ) exposed to 2 . 5 μM ( PR ) 20 for 24 and 48 hours , respectively , with or without heparin ( 0 . 5 μM ) . Nuclei were stained with DAPI ( blue ) . Scale bar ( white ) , 10 μm . ( E , F ) Cell viability quantified CellTiter - Glo luminescence assays mMN ( E ) and NSC34DIFF ( F ) treated as in ( D ) ( n = 3 ) . Data represent mean values ± SEM . Data information : * * , p < 0 . 01 ; * * * , p < 0 . 001 . S

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Figure 7(A) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without 2 μM of a non-coding 38nt oligonucleotide. Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. Note that besides its impact on overall survival, the presence of the oligonucleotide also had a marked effect in rescuing neurite lengths that had been shortened by the peptide. (B,C) Cell viability quantified CellTiter-Glo luminescence assays mMN (B) and NSC34DIFF (C) treated as in (A) (n=3). Data represent mean values ± SEM. ( Data information: **, p<0.01; ***, p<0.001.(D) Immunofluorescence of the neuronal marker TUBB3 (red) in mouse motor neurons differentiated from mESC (mMN) or from NSC34 cells (NSC34DIFF) exposed to 2.5 μM (PR)20 for 24 and 48 hours, respectively, with or without heparin (0.5 μM). Nuclei were stained with DAPI (blue). Scale bar (white), 10 μm. (E, F) Cell viability quantified CellTiter-Glo luminescence assays mMN (E) and NSC34DIFF (F) treated as in (D) (n=3). Data represent mean values ± SEM. Data information: **, p<0.01; ***, p<0.001. S


Figure 1Protein levels of E2F7 and E2F8 during cell cycle progression . HeLa cells were synchronized by a double thymidine block and released into fresh medium . Cells were harvested at the indicated time points and an asynchronous ( AS ) condition was used as control . Protein levels were measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . The asterisk indicates the E2F7 - specific band . Decreased stability of E2F7 and E2F8 in nocodazole - arrested cells . HeLa cells were either treated with DMSO or nocodazole ( 50 ng / ml ) for 16 hours . Cells were harvested and lysed for immunoblotting . Protein expression of cyclin B1 was used as a marker for G2 or M , and γ - tubulin was used as loading control . Selective Cullin - RING inhibitor MLN4924 rescued the degradation of E2F7 / 8 under nocodazole - arrested condition . HeLa cells were treated with DMSO , nocodazole or nocodazole plus MLN4924 ( 0 . 1 μM ) for 16 hours . Cells were harvested and lysed for immunoblotting . cyclin B1 expression was used as a marker for G2 or M cell cycle progression , and γ - tubulin was used as loading control . Increased half - life of E2F7 / 8 by MLN4924 treatment . HeLa cells were either treated with cycloheximide ( CHX , 50 ug / ml ) with or without MLN4924 ( 0 . 1 μM ) . Protein levels of E2F7 and E2F8 were determined by immunoblotting ( left panel ) . Asterisk indicates the E2F7 - specific band . cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression , γ - tubulin was used as loading control . Quantifications ( right panels ) were performed based on two independent experiments . Bar and error bars represent mean ± SEM .

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Figure 1Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.


Figure 2C - terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F . The residues on each motif were mutated to alanines ( R to A , I / L to A ) with site - direct mutagenesis PCR . HEK293 cells were transfected with either EGFP - tagged empty vector ( EGFP ) , wild - types E2F7 / 8 ( WT ) or alanine mutants . Nocodazole ( 50 ng / ml ) was added 32 hours after transfection , and MG132 ( 1 μg / ml ) was added 5 hours before harvesting at 48 hours post transfection . Cells were harvested and lysed for immunoprecipitation using anti - EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP . Schematic view showing the truncated mutants and ML / AA mutant of cyclin F ( Left ) . HEK cells were transfected with indicated constructs and co - immunoprecipitation was performed using Flag resin ( Right ) .

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Figure 2C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.Schematic view showing the truncated mutants and ML/AA mutant of cyclin F (Left). HEK cells were transfected with indicated constructs and co-immunoprecipitation was performed using Flag resin (Right).


Figure 3Wild - type or mutant versions of EGFP - tagged E2F7 / 8 were co - transfected with either empty vector or Flag - tagged cyclin F in HEK293 cells . Nocodazole was added to cells 8h before harvest . 48 hours after transfection , cells were collected and lysed for immunoblotting . Knockdown of cyclin F stabilized E2F7 and E2F8 . HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA . Cells were harvested at 48 hours post transfection . Protein levels of E2F7 / 8 were analyzed by immunoblotting . Asterisk indicates the specific band of E2F7 detection . Cyclin F targets atypical E2Fs during G2 / M . HeLa cells were transfected with either scrambled siRNA ( scr ) or cyclin F siRNA ( sicyclin F ) for 24 hours . Then cells were synchronized by double thymidine block and released into fresh medium after the second block . Cells were harvested at the indicated timepoints after the release . Protein expression was measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . Asterisk indicates the specific detection of endogenous E2F7 . Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo . HEK293 cells were transfected with HA - E2F7 / 8 , with or without Flag - cyclin F , and with HA - tagged ubiquitin . 5 hours before harvest , cells were treated with MG132 . 48 hours after transfection , HEK cells were harvested and lysed for immunoprecipitation pull - down assay with anti - HA resin followed by immunoblotting .

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Figure 3Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection.Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.


Figure 4Schematic view of the experimental setting for the HU - synchronized live cell imaging . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . 16 hours before imaging , cells were synchronized at the G1 / S border by HU ( 2 mM ) treatment . Representative images from different channels are shown , and white arrows in Differential Interference Contrast DIC channel indicate the traced cell . Scale bar : 10µm . Quantification of the number of Ctrl ( left panel ) and 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or sicyclin F that completed mitosis after HU release . For each condition , 100 cells were monitored by live cell imaging . Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance . Schematic view of the experimental setting for live imaging of asynchronous cells . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . At the start of the imaging , G1 cells ( red : mKO2 - Cdt1 > mAG1 - Geminin , 50 cells per condition ) were enrolled and subsequently monitored though their entire cell cycle until mitosis . The black dot represents mitosis . Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8 . Number of Ctrl ( left panel ) or 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown . For each condition , 50 cells at G1 were monitored by live cell imaging . Each cell was followed until it successfully progressed through S and G2 phase , finished mitosis and divided into two daughter cells , for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance . Loss of cyclin F delays the progression from G1 / S transition to mitosis . Histogram shows the time from G1 / S ( mAG1 - Geminin intensity increases to higher than 10 % of the maximum value in three consecutive imaging frames ) to completed mitosis . Only cells that finished mitosis were enrolled in this quantification . Student t - test was used to test the statistical significance , asterisks indicates the P value < 0 . 01 . Bar and error bars represent mean ± SEM . Depletion of cyclin F stalls the cell cycle at late S / G2 . After 24 hours of live cell imaging , the cell cycle progression from panel E was quantified . Histogram shows the percentage of cells at each stage ( at 24h ) over the whole population ( 50 cells per condition ) .

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Figure 4Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.Schematic view of the experimental setting for live imaging of asynchronous cells. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. At the start of the imaging, G1 cells (red: mKO2-Cdt1 > mAG1-Geminin, 50 cells per condition) were enrolled and subsequently monitored though their entire cell cycle until mitosis. The black dot represents mitosis.Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.Loss of cyclin F delays the progression from G1/S transition to mitosis. Histogram shows the time from G1/S (mAG1-Geminin intensity increases to higher than 10% of the maximum value in three consecutive imaging frames) to completed mitosis. Only cells that finished mitosis were enrolled in this quantification. Student t-test was used to test the statistical significance, asterisks indicates the P value <0.01. Bar and error bars represent mean ± SEM.Depletion of cyclin F stalls the cell cycle at late S/G2. After 24 hours of live cell imaging, the cell cycle progression from panel E was quantified. Histogram shows the percentage of cells at each stage (at 24h) over the whole population (50 cells per condition).


Figure 5Disruption of cyclin F binding site increased stability of E2F7 . E2F7WT and E2F7R894A constructs were integrated in to HeLa / TO system . Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium , and cells were collected every 3 hours for immunoblotting . qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels . Bar and error bars represent mean ± SEM . Over - expression of E2F7R894A delays cell cycle progression through G2 - M phase . HeLa / TO cells expressing either wild - type or mutant version of E2F7 were arrested with 16h of HU , and then cells were released into fresh medium with or without doxycycline . Live cell imaging was performed to trace the G2 - M progression of HeLa / TO cells .

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Figure 5Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM.Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.


Figure 6Heatmap showing differentially expressed genes after cyclin F knockdown , and rescued by additional E2F7 / 8 depletion . Highlighted genes are all involved in DNA repair . Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions . qPCR showing the RNA expression of atypical E2F - target genes that are involved in DNA replication or DNA repair . HeLa cells were transfected for 48 hours with siRNAs as indicated . Cells were incubated with nocodazole for 16 hours before harvesting . Data represent averages ± SEM ( n = 3 ) ; * P < 0 . 05 or * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant . Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions . HeLa cells were treated with nocodazole for 16 hours prior to harvesting .

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Figure 6Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.