New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance

Figures

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  FIGURE 1. Platelet C3G regulates ischemia-induced angiogenesis. (A) Representative images of 3 LL tumor sections, from C3G-wt and C3G-KO mice, showing vessel density detected by CD31 staining. Bar: 200 μm. (B) Quantification of the number of vessels per field (0.65 mm2) in tumor sections (Mean ± SEM). (C) Mean ± SEM of the tumor mass (in grams). (D). Mean ± SEM of the percentage of hemagiocytes (CXCR4/VEGFR double-positive cells). (E) Doppler values represented as the mean ± SEM of the percentage of blood flow (n = 6 tgC3G and wtC3G, n = 3 C3G-KO and C3G-wt). (F) Representative Doppler images at the indicated times. The color scale represents the blood flow values. (G) Mean ± SEM of the percentage of CXCR4/VEGFR-double positive cells in blood from C3G-KO and control mice at the indicated times post-ischemia. (H) RT-qPCR analysis of VEGFA, SDF-1 and CD31 mRNA expression in homogenates of murine muscle from the indicated genotypes at day 14 post-ischemia. Values are relative to β-actin expression and were normalized against the corresponding value in the contralateral (non-ischemic) leg. (I) Left: representative images of ischemic leg sections of C3G-wt and C3G-KO mice, showing vessel…

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  FIGURE 2. C3G deletion enhances SDF-1 and VEGF release, while C3G overexpression retains SDF-1. (A) Representative immunofluorescence images of tgC3G, C3G-KO and control platelets, untreated or treated with thrombin (TH, 0.5 U/ml, 5 min) and stained with anti-SDF-1 + Alexa Fluor™-647 (red) and phalloidin-488 (green), taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SD of SDF-1 fluorescence intensity. (B) Representative immunofluorescence images of C3G-KO and control platelets untreated or treated with thrombin (TH, 0.5 U/ml, 5 min) and stained with anti-TSP-1 + Alexa Fluor™-647 (red) or anti-VEGF + Alexa Fluor™-647 (red) and phalloidin-488 (green). All images were taken at the same exposure time. Bar: 2.5 μm. Histograms represent the mean ± SD of VEGF (upper) and TSP-1 (lower) fluorescence intensity. (C) Representative images showing the capillary-like structures formed by HUVECs supplemented with releasates from thrombin-stimulated C3G-KO and C3G-wt platelets. Images were taken every 15 min for 17 intervals. Intervals t0, t7 and t16 are shown, t0 corresponds to 1.5 h after releasate supplementation. Bar: 200 μm. Graphics show the mean ± SEM of different…

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  FIGURE 3. Platelet C3G favors an early onset of melanoma metastasis through regulation of melanoma cell adhesion and TCIPA. (A) Representative flow cytometry plots of lung homogenates from wtC3G and tgC3G animals injected with EGFP-expressing B16-F10 melanoma cells. CT: lung homogenates from untreated mice. Histogram represents the mean ± SEM of the number of EGFP+ cells in 1,000,000 lung cells. (B) Representative sections of lung tissue from tgC3G mice analyzed by immunofluorescence (IF, left) or immunohistochemistry (IHC) with anti-GFP antibodies (right). Presence of B16-F10 cells is indicated with asterisks. Bar: 5 μm. (C) Mean ± SEM of the number of EGFP+ cells in 1,000,000 lung cells from C3G-KO and C3G-wt mice. (D,E) Representative immunofluorescence images of B16-F10 cells adhered to poly-L-Lysine-coated plates, after co-culturing with tgC3G or wtC3G platelets (D) or C3G-KO and C3G-wt platelets (E), stained with anti-P-selectin + anti-goat Cy3 antibodies (to visualize platelets) and phalloidin-488 to detect actin. Arrows indicate the presence of B16-F10 cells. Bar: 50 μm. Histograms represent the mean ± SD of the number of B16-F10 cells per field. (F,I) Mean ± SEM of the number of…

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  FIGURE 4. C3G regulates platelet levels after TPO injection or 5-FU-induced platelet rebound. (A). Line/scatter plots of the mean ± SEM of platelet count in C3G-KO and C3G-wt mice at the indicated time points after TPO injection (n=9 C3G-KO and C3G-wt). (B) Differences in platelet number between two consecutive measurements in each genotype, as an indication of the increased or decreased rate. (C,D) Mean ± SD of the percentage of total (CD41+/CD61+) MKs (C) or mature (CD42+) MKs (D) in BM cultures of C3G-KO and control mice, at the indicated time points. (E,G) Line/scatter plots of the mean ± SEM of platelet count in C3G-KO and C3G- wt mice (E) or in tgC3G and wtC3G mice (G) at the indicated time points after 5-FU-injection (n=8 C3G-KO and C3G-wt; n=12 tgC3G, n=13 wtC3G). (F,H) Differences in platelet number between two consecutive measurements in each genotype, as an indication of the increased or decreased rate. *p < 0.05, **p < 0.01.

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  FIGURE 5. C3G regulates c-Mpl internalization and degradation. (A) Platelets from the indicated genotypes were stimulated with 25 ng/ml (tgC3G lineage) or 100 ng/ml (C3G-KO lineage) TPO for the indicated times and c-Mpl and β-tubulin (β-Tub) or β-actin detected by western blot. Values are relative to β-tubulin or β-actin expression and were normalized against untreated-wild-type platelets or against untreated platelets of each genotype (lower line). (B) Platelets of C3G-KO and control mice were treated 30 min with TPO and stained for c-Mpl surface expression with anti-c-Mpl (AMM2 clone) + anti-rat-Cy5 (Mean ± SD). Representative flow cytometry plots of c-Mpl internalization in each genotype are shown. CT: non-stimulated control. (C) Lysates from C3G-KO and C3G-wt platelets, resting or treated with TPO (100 ng/ml, 5 min were immunoprecipitated with anti-c-Mpl antibody and the levels of ubiquitinated-c-Mpl determined by western blot with anti-ubiquitin antibodies. IP: immunoprecipitation, WB: western blot. L: total cell lysates. IgG and lysate + agarose beads (L+B) were used as negative controls. Values are relative to those in non-stimulated C3G-KO platelets and were normalized to total…

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  FIGURE 6. C3G interacts with c-Cbl and promotes c-Cbl phosphorylation by Src. (A) Lysates from tgC3G platelets, untreated or treated with TPO (100 ng/ml, 5 min were immunoprecipitated with anti-C3G antibodies and the levels of c-Cbl determined by western blot. IP: immunoprecipitation, WB: western blot. IgG and lysate + agarose beads (L+B) were used as negative controls. (B) tgC3G and C3G-KO platelets, and their controls were stimulated with TPO for the indicated time points. Intracellular phospho-c-Cbl levels were determined in fixed cells by flow cytometry, as described (Marklin et al., 2020). Left: representative flow cytometry plots. Right: Histograms represent the mean ± SD of the MFI value, calculated relative to the mean value of untreated wild-type controls. (C) Representative immunofluorescence images of platelets from the indicated genotypes, treated with TPO (100 ng/ml, 5 min and labeled with anti-phospho-c-Cbl + Alexa FluorTM-647 (green) and anti-phospho-Src + Alexa FluorTM-568 (red). All images were taken at the same exposure time. Bar: 2.5 μm. Histograms represent the mean ± SD of the fluorescence intensities of phospho-c-Cbl and phospho-Src. (D) Pearson’s Correlation…

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  FIGURE 7. C3G plays a role in proteasome- and lysosome-mediated degradation of c-Mpl. Platelets from C3G-KO and C3G-wt mice were pre-treated 1 h with 30 μM MG132 (A) or 20 mM NH4Cl (B) prior incubation with 100 ng/ml TPO for the indicated times. The levels of ubiquitinated proteins and c-Mpl were measured by western blot using β-actin as loading control. (C) Histograms represent the quantification of ubiquitinated proteins (left panel) and c-Mpl levels (right panel) relative to β-actin and normalized against the t0 value of C3G-wt. (D) C3G-KO and C3G-wt platelets were treated with 100 ng/ml TPO for 60 min, after 1 h pre-treatment with 30 μM MG132 or 2 mM NH4Cl and labeled with anti-c-Mpl + FluorTM-568 (red) or anti-Ubiquitin + Alexa FluorTM-647 (green). Representative immunofluorescence images of platelets of each genotype under each treatment condition, taken at the same exposure time. Bar: 2.5 μm. The graph shows the Pearson’s Correlation Coefficients (mean ± SD) of c-Mpl and Ubiquitin under the indicated experimental conditions. *p < 0.05, ***p < 0.001.