EMBO/SourceData huggingface dataset:
Figure 6A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I). B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red). Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.G-H. Quantification of CD31+ capillary number per area (mm2; G) and average of capillary cross-sectional area (µm2; H) in Control and CriptoMy-LOF injured muscles at the indicated time points after re-injury (CTX-II) Data are mean±SEM (n=6 biological replicates; *P<0.05; ***P<0.001, Student's t-test).I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).
Figure 7A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury. B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury. Data information: Nuclei were counterstained with DAPI (blue).E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury. F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). Data information: Nuclei were counterstained with DAPI (blue).H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I). Data information: Nuclei were counterstained with DAPI (blue).Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II Data information: Nuclei were counterstained with DAPI (blue).Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF Data information: Nuclei were counterstained with DAPI (blue).Quantification of VEcad/KLF4 double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-IIQuantification of VEcad/KLF4 double positive cells per area (mm2) in diaphragm sections of mdx-Control and mdx-CriptoMy-LOF
Figure 1A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5.D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4).E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3.G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3.H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.
Figure 2A Western blot analysis of autophagy markers, Beclin1, ATG3, ATG7, ATG12-5, and LC3I/II, and the mitophagy-related proteins Parkin and PINK1 in HC (#1-3) and DM platelets (#1-8).B Quantification of autophagy markers and mitophagy-related proteins from HC (#1-5) and DM (#1-18) individuals. (Beclin1; **p=0.0003, ATG12; *p=0.0388, ATG3; *p=0.0213, ATG7; p=0.4051, LC3; *p=0.0298, PINK1; *p=0.0155, Parkin; *p=0.0103 vs. HC). GAPDH served as a loading control.C The three panels represent autophagy stages found in DM platelets. Autophagosome structures containing/enclosing organelles are indicated by the black box. Shown for each panel is an enlargement of the autophagosome (DM1 and 2) or the autolysosome (DM3). Dashed structures are an outline of the autophagosome membranes.D LC3 immuno-EM analysis of HC and DM platelets. Arrows indicate immunogold-labeled LC3 clusters. No clusters were found in HC platelets. Representative areas of clusters of gold labeling in DM patients (DM1-3) are presented. Shown in the insets are enlargements of the gold clusters adjacent to mitochondria-like structures.E Double staining for MitoTracker (mitochondria membrane potential) (50nM for 15 min) and LC3 (autophagy) in HC and DM platelets. Boxed platelets are enlarged in Panel F.F A typical HC platelet has high membrane potential and low LC3 in contrast to a representative DM platelet which has low membrane potential and high LC3.G Quantification of autophagosome positive platelets using 6 HC and 4 DM samples after staining using CoxIV (mitochondria) and LC3 (autophagy marker). The y-axis indicates percentage of LC3 positive cells in total cells (**p=0.006 vs. HC).
Figure 3A Representative western blot analysis of recognized autophagy signaling molecules (pJNK, JNK, pAKT, and AKT, pmTOR, and mTOR) in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis of autophagy upstream signaling molecules on HC (#1-5) and DM (#1-18) individuals. (pAKT/AKT; p=0.0747, pJNK/JNK; *p=0.001, pmTOR/mTOR; p=0.3356 vs. HC). GAPDH was used as the loading control.B Representative western blot analysis using HC and HG platelets with or without H2O2. And assessing for LC3I/II. GAPDH was used as the loading control.C Platelet suspensions were incubated in HG for 2 hours and with H2O2 (1mM). ROS levels were evaluated using a detection kit (Enzo Life Science) for 60 mins at 37 ºC.D Western blot analysis of p53, JNK and LC3I/II in HC platelets treated with H2O2 (1mM for 1 hour) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control.E Confocal microscopy was used to corroborate the Western analysis using triple staining for Mitotracker, CoxIV, and LC3 in HC or H2O2. The insets highlight the association of low membrane potential with high levels of LC3 and increased colocalization of Cox IV with LC3 in H2O2-treated platelets.F HC platelets were pretreated with H2O2 to induce autophagy and then treated with or without SP600125 (JNK inhibitor) at 3 concentrations (1, 5 and 10 µM) to assess for inhibition of autophagy. This Western blot is representative of three independent experiments. Quantification of the three independent experiments is provided at right. (pJNK/JNK in H2O2/SP600125; **p=0.0286 vs. H2O2 group, LC3II in H2O2; *p=0.0241 vs. HC group, LC3II in H2O2/SP600125; **p=0.0044 vs. H2O2 group, n=3 for each group).G Triple staining of CoxIV, LC3, and LAMP1 in HC and H2O2 with/without SP600125-treated platelets using confocal microscopy. Arrow indicates localization of LC3 and LAMP1. Graph indicates colocalization between LC3, LAMP1 and CoxIV signal. The y-axis indicates fold change of colocalization between LC3 and LAMP1 in the mitochondria area. Signal intensity of each group was converted to fold and compared with HC values (H2O2;*p=0.023 vs. HC, H2O2/SP600125;*p=0.04 vs. H2O2 group).
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)
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.
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).
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, 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)
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 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 shown