EMBO/SourceData huggingface dataset:
Figure 1E Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function in GA of mice receiving PBS or indicated EVs. Data information: Western blot signals are quantified and normalized to Gapdh.F RT-qPCR showing relative levels of miR-122 and indicated mRNAs in GA of indicated groups of mice. Data were normalized to U6 (for miR-122) or Ppib (for all mRNAs) and compared to control group receiving PBS (one-way ANOVA, n=4 or 5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.H ROS levels in GA from indicated groups of mice detected by DHE staining (red). DAPI (blue) shows the nuclei. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.I ROS levels in GA from mice that had received indicated EV treatment for 5 weeks. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.J RT-qPCR showing relative levels of indicated mRNAs in GA of indicated groups of mice (one-way ANOVA, n=5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.K Relative mtDNA/nDNA ratio in GA determined by PCR (one-way ANOVA, n=5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.
Figure 2C Responsiveness of the reporters to miR-122 mimic in transfected C2C12 (t-test, n=3 biological replicates). Data information: In bar graph, values are shown as mean ± SD. **P<0.01, ns: not significant.D CFSE signals in C2C12 myotubes treated with indicated CFSE-labeled Evs for 24 h, indicating EV uptake. Scale bar: 100 µm. Data information: In bar graph, values are shown as mean ± SD. **P<0.01, ns: not significant.
Figure 3A Western blot showing levels of indicated proteins in C2C12 treated with indicated EVs, or transfected as indicated with miR-122 mimic or TP53 expression plasmid carrying silent mutation of the miR-122 binding site. Data information: Western blot signals are quantified and normalized to Gapdh.C Relative ATP levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.D Relative ROS levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3-5 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.
Figure 4B Indicated MDA-MB-231-derived BC cells were injected into the mammary fat pad of female NSG mice to form tumors. Tumor-bearing and age-matched tumor-free mice were sacrificed for muscle collection at week 6. Western blots show protein levels in GA. Western blot signals are quantified and normalized to Gapdh.D RT-qPCR indicated mRNAs (D) in GA of tumor-free and tumor-bearing mice. Data were normalized to Ppib (for all mRNAs) and compared to the tumor-free group (one-way ANOVA, n=4 or 5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.F Representative TEM images showing SS and IMF mitochondria and their quantification in GA (one-way ANOVA, n=3 mice per group for the left subpanel and n=154-164 mitochondria analyzed per group for the right subpanel). Scale bar: 1 µm. Yellow bars: depths of SS mitochondrial areas; arrows: IMF mitochondrial area. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.H Western blots of GA from female BALB/c mice bearing 4T1-derived tumors with tumor-free mice as controls. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. Western blot signals are quantified and normalized to Gapdh.I RT-qPCR showing levels of indicated genes in of GA from indicated BALB/c mice (one-way ANOVA, n=4 or 5 mice). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.
Figure 5A GA were collected from female NSG mice that had received indicated EVs or PBS for 5 weeks. Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function. Data information: Western blot signals are quantified and normalized to Gapdh.B Relative mtDNA/nDNA ratio in GA determined by PCR (one-way ANOVA, n=4 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ns: not significant.C RT-qPCR showing indicated mRNAs in GA of indicated groups of mice. Data were normalized to Ppib and compared to control group receiving PBS (one-way ANOVA, n=4 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ns: not significant.
Figure 6AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. A Western blot showing indicated protein levels in the GA. Western blot signals are quantified and normalized to Gapdh.AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. C Relative mtDNA/nDNA ratio in GA determined by PCR (two-tailed Student's t-test, n=8 mice per group). Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. D Representative TEM images showing SS and IMF mitochondria and their quantification in GA (two-tailed Student's t-test, n=3 mice per group for the left subpanel and n=166 or 181 mitochondria analyzed per group for the right subpanel). Scale bar: 1 µm. Yellow bars: depths of SS mitochondrial areas; arrows: IMF mitochondrial area. Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. E ROS levels in GA from indicated groups of mice. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (two-tailed Student's t-test, n=4 mice per group). Scale bar: 50 µm. Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.
Figure 1D. Representative genotyping of the c.1192C>A mutation by allele-specific PCR. For each sample, two PCRs were performed, one with a specific primer for the 1192C allele (PCR 1) and a second with specific primers for the 1192A allele (PCR 2). An 821-bp band indicated the presence of the allele; amplification failure indicated the absence of the allele. The 434 bp amplification product of the hGH control primer is presented in all lanes. In the gel, the first lane (MW) shows the 100 bp DNA ladder marker ; for the other lanes, C and A indicate the product from PCR 1 and PCR 2, respectively, amplified from RIF+/+ (1(CC)), RIF+/- (2(CA)) and RIF-/- (3(AA)) samples.
Figure 2A and B. Flow analysis of SLC44A2 expression at the surface of RBCs from control donors (red), RIF- proband 1 (blue), and VER- proband 2 (green) using anti-RIF (A) and anti-VER antibodies (B). The light gray profile corresponds to RBCs incubated with only the secondary antibody.
Figure 3B. Western blotting shows that the anti-RIF and anti-VER antibodies bind to HuSLC44A2. Western blots of whole Sf9 lysates (WCL) expressing full-length recombinant human SLC44A2 cDNA (rHuSLC44A2) were probed with RIF and VER antisera (1:10). Lanes 1 and 2 show strong binding to rHuSLC44A2. Titer differences in the sera required longer exposure (10 min) of the filter to the film to develop the image with VER serum compared to only 30 second exposure for RIF serum. A similar blot of immunoprecipitated rHuSLC44A2 from the Sf9 lysates similarly reacted with RIF and VER sera (lanes 3 and 4) showed equivalent exposure times (30 seconds for both sera) when tested against the concentrated rHuSLC44A2 protein.C. The anti RIF and VER antibodies bound to immunoprecipitated wild-type SLC44A2 in lysates of the UM-SCC-47 human squamous cancer cell line (exposure time 5 min).D. Western blot of murine Slc44a2 protein immunoprecipitated from wild-type and Slc44a2 knockout mice with rabbit anti CTL2-NT serum was probed with anti CTL2-NT (left panel) and with VER serum (center Panel) or RIF serum (right panel). The left panel was developed with goat anti rabbit IgG heavy and light chain ; the center and right panels were developed with rabbit anti human IgG and IgM heavy and light chain specific
Figure 4A. CT angiography of the giant unruptured intracranial aneurysm on the right posterior cerebellar artery (PCA, blue arrow).B. 3D volume rendered of multiple cerebral aneurysms on the right PCA (a), on right posterior communicating artery (b), and on right middle cerebral artery (c).C and D. Angiograms before and after embolization of the PCA aneurysm, respectively.
Figure 5A. Terminal differentiation of HSPCs from healthy control and SLC44A2null proband was monitored via α4-integrin and band 3 levels of GPApos cells at day 6 after EPO addition.B. Cell proliferation during in vitro erythropoiesis.
Figure 6A. SLC44A2 transcript variants P1 and P2 were amplified from cDNA samples isolated from control reticulocytes (CTRL RETIC), neutrophils (CTRL NEUTRO), and PBMC of the VER- proband 2 (VER PBMC). The purity of reticulocytes and neutrophils was verified by flow cytometry using anti-CD71 and anti-CD16 antibodies, respectively.D. Effect of anti-VER and anti-RIF on the adhesion of neutrophils from healthy controls to endothelial cells (HMEC-1). Adherent neutrophils were quantified and means ± standard error of the median of 3 independent experiments are shown. **P < .01, ***P < .001, ****P < .0001. Statistical analyses were performed using GraphPad Prism 7. Statistical test was performed using paired non-parametric t-test (Wilcoxon t-test).
Figure 1B-E (B, D) Representative microscopy images of the mitochondrial network morphology in neurons of the amygdala (B) and the CA1 region of the hippocampus (D) in mitoCFP+/P301L- mice and mitoCFP+/P301L+ mice. MitoCFP signal is displayed in gray on the images. Gray scale bars: 5 µm. (C, E) Mitochondrial length in neurons of the amygdala (C) and in Ca1 (E) of mitoCFP+/P301L- and mitoCFP+/P301L+ mice. Data information: Values represent the mean ±SEM of n=11 MitoCFP+/P301L- mice and n= 13 MitoCFP+/P301L+ mice. Each gray open circle represents the mean of 6 technical replicates (data from 6 images) per animal. * P<0.05, ** P<0.01, Student unpaired t-test.F-I (F, H) Representative microscopy images of the endoplasmic reticulum stained with calnexin (in red) and mitochondria (mitoCFP) in neurons of the amygdala (F) and Ca1 (H) in mitoCFP+/P301L- and mitoCFP+/P301L+ mice. (G, I) Colocalization analysis between calnexin and mitoCFP in neurons of the amygdala (G) and Ca1 (I) of mitoCFP+/P301L- and mitoCFP+/P301L+ mice. The Manders' overlap coefficients M2 is represented on the graphs, as it indicates the proportion of overlaps between the mitoCFP signal (channel 2) and calnexin signal (channel 1) relative to the total pixel intensity of channel 2 (i.e. only in neurons, as mitoCFP is expressed only in these cells). Data information: Values represent the mean ±SEM of n=11 MitoCFP+/P301L- mice and n= 13 MitoCFP+/P301L+ mice. Each gray open circle represents the mean of 6 technical replicates (data from 6 images) per animal. * P<0.05, ** P<0.01, Student unpaired t-test.
Figure 3G Quantification of total cholesterol levels in cell lysates from WT, P301L and Tau KO cells. Data information: (G Data are presented as mean ±SEM of n = 12-18 replicates per group (4 independent experiments) in percentage of the WT cells. * P<0.05, ***P<0.001; One-way ANOVA + Tukey's post hoc test.K-N Mitochondrial cholesterol levels (K, L) and pregnenolone levels (M, N) in WT, P301L and Tau KO cells (K, M) and in brain preparations from WT and pR5 mice (L, N). Data information: K, M) Data are presented as mean ±SEM of n = 12-18 replicates per group (4 independent experiments) in percentage of the WT cells. * P<0.05, ***P<0.001; One-way ANOVA + Tukey's post hoc test. L, N) Data are presented as mean ±SEM of n = 6 WT and n = 5 pR5 mice with 2 technical replicates per animal. * P<0.05, ** P<0.01, Student unpaired t-test.O-Q Quantification of total cholesterol levels (O), mitochondrial cholesterol levels (P) and pregnenolone levels (Q) in patient-derived induced pluripotent stem cells (iPSC) bearing a P301L tau mutation (iPSC-P301L) and the corresponding isogenic wild-type cells (iPSC-WT). (O-Q) Data are presented as mean ±SEM of n = 27 replicates/group (O), n = 9 replicates/group (P) and n = 18 replicates/group (Q). * P<0.05, *** P<0.001, Student unpaired t-test.
Figure 5A Visualization of the VAPB-PTPIP51 interactions by in situ proximity ligation assays (PLAs) in wild-type (WT) cells and P301L cells transfected with the control (Ctrl) siRNA, as well as in MFN2 siRNA transfected cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. White scale bars: 20 µm B Quantitative analysis of the PLA signal represented as number of contact points between VAPB-PTPIP51 per cell in percentage of the WT cells. In total, 60 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ± SEM. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B.C-D Representative microscopy images (z-projections) of co-staining with Filipin III in green and (C) TOMM20 (mitochondria) or (D) ER-RFP (endoplasmic reticulum) in red with xy- and xz-axis orthogonal views framing the corresponding image. White scale bars: 10 µm Data information: * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20E-H Manders' coefficient M1 representing the proportion of Filipin III overlapping with (E) TOMM20 or (G) ER-RFP, and M2 representing the proportion of (F) TOMM20 or (H) ER-RFP, overlapping with Filipin III. In total, 20-60 cells were analyzed per group (4 independent experiments). Data information: E-J) Data are presented as mean ± SEM. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein; TOMM20: translocase of the outer mitochondrial membrane complex subunit 20;
Figure 6A-D (A, C) Representative microscopy images (z-projections) of tau phosphorylation using (A) AT8 and (C) AT180 antibodies (in red) in GFP-tagged P301L cells (GFP signal in gray) treated for 24 h with CHIR99021 (CHIR) (right) and untreated cells (left). White scale bars: 25 µm. (B, D) Quantification of (B) AT8 and (D) AT180 staining fluorescence intensity. Data are presented as mean ±SEM of n = 50-60 cells per group (4 independent experiments). *** P<0.001; Student unpaired t-test. Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells.E Visualization of the VAPB-PTPIP51 interactions by in situ proximity ligation assay (PLA) in untreated wild-type (WT) cells and P301L cells as well as in CHIR-treated cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. White scale bars: 20 µm F Quantitative analysis of the PLA signal represented as number of contact points between VAPB-PTPIP51 per cell in percentage of the WT cells. In total, 60 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B.G-H Representative microscopy images (z-projections) of co-staining with Filipin III in green and (G) TOMM20 (mitochondria) or (H) ER-RFP (endoplasmic reticulum) in red with xy- and xz-axis orthogonal views framing the corresponding image. White scale bars: 10 µm Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20I-L Manders' coefficients M1 representing the proportion of Filipin III overlapping with (I) TOMM20 or (K) ER-RFP, and M2 representing the proportion of (J) TOMM20 or (L) ER-RFP, overlapping with Filipin III in untreated WT cells and P301L cells as well as in CHIR-treated cells. In total, 35-75 cells were analyzed per group (4 independent experiments). Data information: Data are presented as mean ±SEM of n = 50 replicates per group (5 independent experiments) in percentage of the WT cells. P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. ER-RFP: endoplasmic reticulum red fluorescent protein TOMM20: translocase of the outer mitochondrial membrane complex subunit 20;
Figure 1A Immunostaining of a murine knee joint at 18.5 dpc with an anti GCP-2 or IgG control antibody.B Immunohistochemistry for GCP-2 (red) in the knee of a human 5-month old fetus.C, D Immunostaining for GCP-2 in an adult (12 week old) mouse knee (C) or in adult human articular cartilage (D).
Figure 2GAG content as assessed by Alcian blue staining with densitometry (A) A HAC micromasses treated with a GCP-2 neutralizing antibody (n=4) or IgG control (n=4); p values were determined by the Mann-Whitney U test.GAG content as assessed by Alcian blue staining with spectrophotometric B C28/I2 micromasses treated with GCP-2 siRNA (n=12) or scrambled control siRNA (Scr; n=14); p values were determined by unpaired two-tailed Student's t test.GAG content as assessed by Alcian blue staining spectrophotometric C C3H10T½ micromasses treated for 3 days with recombinant GCP-2 100 ng/ml (n=4) or vehicle (n=4) or BMP-2 (n=4); p values were determined by ANOVA followed by Tukey HSD post hoc test.D qPCR for ACAN (Aggrecan) expression in C3H10T½ micromasses treated for 3 days with recombinant GCP-2 (100 ng/ml) (n=3) or vehicle (n=4); ACAN - Aggrecan;E spectrophotometric quantification of GAG release (dimethylmethylene blue assay) in supernatant of human cartilage explants treated with recombinant GCP-2 (n=5) or vehicle (n=5) and incubated for 24hF Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co-implantation of HACs in a 10:1 ratio with growth-arrested COS7 expressing either GFP (n=3) or GCP-2 (n=3) and quantification - on the right-hand side - of the metachromatically stained area as % of total area; scale bar =200µm;G Collagen 2 (Col2) immunostaining (left) and quantification (right) as % of total area; scale bar = 100µm; n = 3 per group; p values were determined by unpaired two-tailed Student's t test.
Figure 3A, B qPCR for (A) Runx2 and (B) Col1A1 expression in HAC micromasses treated for 24 hours with recombinant GCP-2 (100 ng/ml) or vehicle (n=4); Runx2 = Runt-related transcription factor 2, Col1A1 = collagen type 1 alpha 1; n=4C Alkaline phosphatase staining (red) in HAC monolayer after 3 week treatment with T3 hormone (100 ng/ml), T3 hormone + GCP-2 (100ng/ml), or control medium; scale bar 100µm.D quantification of alkaline phosphatase stained area (n=3); p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.E qPCR for Col10A1 (n=3) of HAC 3 weeks after treatment with vehicle, T3 (100 ng/ml) or T3+GCP-2 (100 ng/ml); n=3;F, H C3H10T½ monolayers were treated with osteogenic medium (OM) or OM + GCP-2 (100 ng/ml): (F) number of cells per well positive for alkaline phosphatase staining (n=3 in OM and n=4 in OM+GCP-2), (G) number of alkaline phosphatase positive nodules per well (n=7), and (H) spectrophotometric quantification of alizarin red staining at 570nm (n=6)