EMBO/SourceData huggingface dataset:
Figure 1B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals.C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right).D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher's exact test.E. Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.
Figure 2A-B. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test.C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).
Figure 3A. Expression of netrin-1 and DCC in lymphoma cell lines. Quantification was performed by Q-RT-PCR in 21 lymphoma cell lines. HPRT housekeeping gene was used as a standardization control. Netrin-1 and DCC levels are indicated as follow: −, not detectable; + to ++++, moderate to very high expression. ABC and GC-DLBCL cell lines are highlighted respectively in blue and green, MCL ones are in grey. Expression of netrin-1 (in green) by immunofluorescence using netrin-1 antibody on OCI-Ly3 and Granta-519 cell lines is shown on the right panel. Nuclei are counterstained in blue by Hoechst.B. Caspase-3 activity in Granta-519 cells after transfection of scrambled siRNA (si-Ctl) or netrin-1 siRNA (si-netrin-1), with or without DCC siRNA (si-DCC). Results are means+/-std of four independent experiments. $: p=0.03; *: p=0.02; two-sided Mann-Whitney U-test.D. Effect of net-1 mAb on Granta-519 (left panel) and OCI-Ly3 (right panel) cell density. Results are means+/-std indexed to control of four independent experiments. $: p=0.05; *: p=0.03; two-sided Mann-Whitney U-test.E. Effect of net-1 mAb on induction of Granta-519 cells apoptosis, detected by TUNEL staining. Left panel: representative images are shown. TUNEL positive cells are labelled in red. Nuclei are counterstained in blue by Hoechst staining. Right panel: quantification of one representative experiment out of three performed is exposed. Results are presented as percentage of TUNEL-positive cells per field. *: p=0.04; two-sided Mann-Whitney U-test.F. Caspase-3 activity in Granta-519 cells treated with net-1 mAb antibody or with an unrelated Ig-G1 antibody (Ctl), with or without silencing of DCC via siRNA (siDCC). Results are means+/-std of four independent experiments. $: p=0.05; *: p=0.02; two-sided Mann-Whitney U-test.G-H. Effect of netrin-1 interfering antibody net-1 mAb on OCI-Ly3 (G) and Granta-519 (H) xenograft tumors growth. Results are presented as means+/-95% CIs of tumor growth rates from the beginning of treatment. *:p=0.02; **:p=0.01; $: p=0.007; $$:p=0.002; ***: p=0.0002; ****:p<0.0001; two-sided Student t-test (Granta-519) or Welch t-test (OCI-Ly3).I. Caspase-3 activity in tumors from control (n=4) or netrin-1-interfering antibody (n=4) treated mice. Tumors resection was performed at the end of the treatment. Results are means +/-std of caspase-3 activity. *:p=0.02; two-sided Mann-Whitney test.
Figure 1Electron microscopy showing two adjacent mitochondria (one intact and one ruptured) in a DM platelet. a. Lower powered view of the single DM platelet. b. The enlarged inset highlights the ruptured mitochondria (with loss of cristae morphology) next to the intact mitochondria. c. Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation. (M- mitochondria)Immunoprecipitation (Meng et al) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I/II western.LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15 nm dots indicate immunogold-labeled LC3 clusters and 5nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b-d) are presented. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 2Western blot analysis of MsrB2 and LC3 I/II in Meg-01 cell after shMsrB2 transfection (72 hrs). Cells were then treated with H2O2 (1 mM for 1 hr) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control. (shMsrB2 vs. shCon; *p=0.024, shMsrB2/H2O2 vs. shCon/H2O2; *p=0.0295, shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC **p=0.0100, n=3)Western blot analysis of MsrB2, pp53(S15) and LC3 I/II in Meg-01 cell after 25 mM HG treatment for 24 hrs. GAPDH was used as the loading control. Quantification analysis on individual groups. GAPDH served as the loading control. (MsrB2: shCon/HG vs. shCon; **p=0.0017, shMsrB2/HG vs. shCon/HG; *p=0.0189, pp53: shMsrB2/HG vs. shCon/HG; *p=0.0317, LC3II: shCon/HG vs. shCon; *p=0.0215, shMsrB2/HG vs. shCon/HG; *p=0.0158, n=3) The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 3Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA. (ROS; *p=0.0139, MsrB2 fl/fl n=5, MsrB2 -/- n=6)C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice. Results are expressed as % apoptotic cells. (TMRE; *p=0.0118, MsrB2 fl/fl n=5, MsrB2 -/- n=5)Representative images showing the intraplatelet expression of LC3 and active caspase-3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest. Magnification: 100X. Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase-3.Graph indicates platelets positive for active caspase-3 signal. The y‐axis indicates percentage of caspase-3 positive cells from total cells calculated from n = 10 independent images per sample. (active caspase-3 signal ; **p=0.0003Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n=6 samples per group. Values expressed as Mean ± SEM. (caspase-3 activity ; *p=0.0157)and H. Western blot analysis of Parkin, LC3I/II, MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice. Control peptide (CP), LIF peptide (Foroud et al). Quantification analysis of individual band intensity. (Parkin; *p=0.02964, LC3II; *p=0.03200, MsrB2; **p=0.0059, Active caspase3; *p=0.0368 vs. chow or HFD group, each group n=3). Actin served as the loading control. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 4Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets, followed by western blot analysis of precipitated Parkin and modified Parkin.Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and MsrB2.Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin, LC3 and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of Parkin, LC3 and MsrB2 as determined by Volocity software (PerkinElmer, USA). Graphical quantification of colocalization between Parkin, LC3 and MsrB2 signal.Signal intensity of each group was converted to fold and compared with HC values (colocalization of parkin, LC3 and MsrB2; **p=0.0006).Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin, was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer.In vitro assay. Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs. Western blot analysis of Parkin and MsrB2 was performed. (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 5Western blot analysis of Methionine Sulfoxidation (MetO), Methionine sulfide reductase B2 (MsrB2) and Parkin in human Healthy Control (HC) and Diabetic Mellitus (DM) platelets. P.C. is a BSA-MetO positive control (Cayman Chemical Co.). Quantitation of western blot analysis shown in Fig 1C. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (MetO; **p=0.0003, MsrB2; *p=0.0186, Parkin; **p=0.0003 vs. HC).Western blot analysis of Parkin and Parkin aggregation in human Healthy Control (HC) (n=3) and Diabetic Mellitus (DM) (n=12) platelets. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (modified parkin *p=0.035 vs. HC). Actin served as the loading control.Parkin IP was performed in HC, HC plus H2O2, and DM patients.MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 6Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets. Western blot analysis of precipitated MsrB2, Parkin and ubiquitinated MsrB2 (UB).Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV (mitochondria marker), ubiquitin and MsrB2. Colocalization is demomstrated by arrows and quantitated. (colocalization ubiquitin, MsrB2; **p=0.0003 vs. HC).MsrB2 ubiquitination assay after transient transfection of MsrB2-GFP (1µg) with RFP empty vector (E.V.) or RFP-Parkin (3µg) in HEK293. After transfection (48hrs), an IP was performed using GFP-Trap bead, followed by Western blot analysis using UB and MsrB2 and Parkin antibodies. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 7Western blot analysis of MsrB2 and Cytochrome C in isolated mitochondria after incubation w/o Cyclosporin A (CsA; 2 μM for 2 hrs.) or w/ CsA. ATPB served as the loading and negative control (mitochondrial matrix protein).Western blot analysis of Parkin, LC3I/II, ubiquitinated MsrB2 in chow (n=4) and HFD (n=6) mouse platelets.Confocal microscopy was initially stained with Tubulin antibody (Panel a., Chow diet, or b. High Fat Diet) to assess platelet architecture. This was followed by staining using ATPB (mitochondria marker - green) and MsrB2 antibodies (Blue) (Panel c-e). There was loss of colocalization in the HFD mice supporting release (blue) from ruptured mitochondria (green) (Panel d & e). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 8Representative western blot analysis of MsrB2, LC3I/II and parkin using Parkinson's disease (PD) platelets. Quantification analysis of individual band intensity. (MsrB2; *p=0.0255, LC3II; *p=0.0101 vs. HC (n=5) and PD (n=12)). GAPDH served as the loading control.Platelet apoptosis (Annexin V) were measured by flow cytometry analysis in HC (n=9) and PD (n=13) platelets. (**p<0.01 vs. HC). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.
Figure 1A-F Indirect calorimetry was performed using the Oxymax/CLAMS system in Cdk4+/+ (n=6) and Cdk4-/- (n=5) mice. Whole-body oxygen consumption rate (VO2) (A-B), energy expenditure (EE) (C-D) and respiratory exchange ratio (RER) (E-F) were measured at 24°C and during a cold challenge at 6°C during both the light (white rectangle) and dark (black rectangle) phases over a 2-day period.CalR was used to GLM-regression plot for each group corresponding to the association between energy balance and total body mass at 24°C (G) and the association between energy balance and total body mass at 6°C (H).J-K Basal rectal temperature was measured at 24°C (J), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (K) in Cdk4+/+ (n=10) and Cdk4-/- (n=8) mice.
Figure 2A Gross morphology of iBAT from Cdk4+/+ and Cdk4-/- mice.C Hematoxylin-eosin staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm) from Cdk4+/+ and Cdk4-/- mice.D-E Western blot analysis of UCP1 and CDK4 protein expression (D) and quantified protein levels of UCP1 (E) (n=4 biological replicates) in iBAT. HSP90 was used as loading control.F Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4+/+ (n=6) and Cdk4-/- (n=6) mice as assessed by RT-PCR.G-H Transmission electron microscopy (TEM) images of iBAT (G) (scale bar 5 μm (upper panel) and 1 μm (lower panel, LD indicates lipid droplet and M mitochondria)) and quantification of lipid droplet number (LD) and mitochondrial volume (Mito) (H) (Cdk4+/+ (n=4) and Cdk4-/- (n=4)).I Respirometry analysis of iBAT as measured by the Oroboros system (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).
Figure 3A-B Rectal temperature was measured at 24°C (A), and rectal temperature was monitored for 6 hours during acute cold exposure at 4°C (B) in Cdk4flox/flox (n=9) and Cdk4flox/flox Ucp1-Cre mice (n=9).C Gross morphology of iBAT from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice D iBAT mass compared to body weight of Cdk4flox/flox (n=8) and Cdk4flox/flox Ucp1-Cre (n=7) mice. E Hematoxylin-eosin (H&E) staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm (upper panel) and 50 μm (lower panel)) from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice.F-G Western blot analysis of UCP1 and CDK4 protein expression (F) and quantified protein levels of UCP1(G) (n=4 biological replicates) in iBAT. HSP90 was used as a loading control.H Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4flox/flox (n=5) and Cdk4flox/flox Ucp1-Cre (n=6) mice as assessed by RT-PCR.
Figure 4A-B TH immunohistochemical staining in iBAT sections (A) (scale bar 20 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (B) (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).C Hematoxylin-eosin staining in scWAT showing multilocular adipocytes in Cdk4-/- (scale bar 50 μm).D Expression of thermogenic genes and brown adipocyte markers in scWAT of Cdk4+/+ (n=5) and Cdk4-/- (n=6) mice as assessed by RT-PCR.E-F Acute cold test (4°C) after treatment with the β-adrenergic antagonist propranolol (Prop) in Cdk4+/+ (n=7) and Cdk4-/- (n=6) animals or with vehicle (NaCl) (Cdk4+/+ (n=7) and Cdk4-/- (n=6)) (E) and corresponding quantification of the area under the curve (AUC).
Figure 5A Cdk4 mRNA expression in the dissected VMH of Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre (n=6) miceB-C Rectal temperature was measured at 24°C (B), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (C) in Cdk4flox/flox (n=15) and Cdk4flox/flox Sf1-Cre mice (n=15).D Hematoxylin-eosin (H&E) staining of iBAT sections (scale bar 100 μm) from Cdk4flox/flox and Cdk4flox/flox Sf1-Cre mice.E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox/flox (n=9) and Cdk4flox/flox Sf1-Cre mice (n=9) mice as assessed by RT-PCR.F-G TH immunohistochemical (IHC) staining in iBAT sections (F) (scale bar 40 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (G) (Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre mice (n=5)).H-I Western blot analysis (H) and quantification of p-CREBS133 protein expression following 6 hours of cold exposure (4°C) (I) (n=3 biological replicates). HSP90 was used as a loading control.
Figure 6A-D Confocal illustrations of frontal hypothalamic sections from Cdk4+/+ mice (A, C, N=3) and Cdk4-/- mice (B, D, N=3). Low magnification showing nuclei (Hoechst, left pannel) and c-Fos activated-cells (white, right pannel) in the mPOA (A1-2, B1-2) and the ARC, the VMH, the DMH, the LH (C1-2, D1-2). High magnifications corresponding to the frame area in A1-2, B1-2, C2, D2 and showing c-Fos-IR cells (A3-4, B3-4, C3-6, D3-6). Scale bars (A1-2, B1-2, C1-2, D1-2): 200μm, (A3-4, B3-4, C3-4, D3-4): 50μm. E Quantification of c-Fos IR cells in brain sections of Cdk4+/+ and Cdk4-/- mice (n=15-24 slices according hypothalamic nucleus and genotype).
Figure 1A, B. A fraction of OTULIN is localized at mitochondria where it suppresses M1-linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1-linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used.C. TNF induces M1-ubiquitination at mitochondria in various cell types. The indicated cell types were treated with TNF (25 ng/ml, 15 min) and purified mitochondrial fractions analyzed as described in A.D. TNF-induced mitochondrial M1-ubiquitination does not occur in HOIP-deficient cells. Wildtype (WT) and HOIP-KO HAP cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.E. Mitochondria and M1-linked ubiquitin co-localize after TNF treatment. SH-SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1-ubiquitin (1E3, red) and analyzed by SR-SIM.F. TNF induces recruitment of LUBAC components to mitochondria. HEK293T cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.G. TNF induces a fast and transient increase in M1- and K63-specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1-, K63-, and K48-specific ubiquitin antibodies.
Figure 2A, B. PINK1 is stabilized by TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) before harvesting or incubated with OTULIN-specific siRNA for 48 h (A). Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies. Quantification of PINK1-specific signals normalized to TIM23 is shown in the right panel. Data represent the mean values with standard deviations of 4 independent experiments. *p < 0.05. A two-tailed non-parametric Mann-Whitney U-test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1.C. TNF-induced PINK1 stabilization does not induce mitophagy. HeLa cells expressing mt-mKeima were treated with TNF (25 ng/ml) for 30 min or 16 h. As a control, mt-mKeima -expressing Hela cells were treated with antimycin A and oligomycin (A/O, 10 µM each) for 1 h. The analysis was done by flow cytometry gating of lysosomal and neutral mt-mKeima.D. Catalytically active HOIP ubiquitinates overexpressed PINK1. HEK293T cells were transfected with the plasmids indicated. After 24 h, the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin.E. Recombinant HOIP ubiquitinates PINK1 in vitro. V5-tagged-PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1, UBE2L3, C-terminal HOIP and ubiquitin for in vitro ubiquitination. The samples were then analyzed by immunoblotting using V5 antibodies.F. Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min), then endogenous PINK1 was immunoprecipitated from mitochondrial fractions. Cells mildly overexpressing PINK1-V5 were also included (lanes 4, 5, 8), to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1. As a control for the presence of M1-linked ubiquitin chains, immunoprecipitated PINK1 was treated with recombinant OTULIN. As controls for the specificity of the immunoprecipitation, beads only (lanes 6 - 8) and beads plus IgG (lane 9) were included. The samples were analyzed by immunoblotting using antibodies against PINK1, M1-ubiquitin, and phosphorylated ubiquitin. For immunoprecipitation of overexpressed PINK1, only 50% of cells were used in comparison to the immunoprecipitation of endogenous PINK1.