DataSheet1_New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance.pdf
Table S1. Genetically modified animals. Table S2. PCR primers. Table S3. Antibodies used for flow cytometry, confocal immunofluorescence microscopy, immunohistochemistry, immunoprecipitation and western blot. Table S4. Deletion of C3G did not modify platelet counts and its parameters. Platelet numbe...
| Autores: | , , , , , , , |
|---|---|
| Formato: | conjunto de datos |
| Estado: | Versión publicada |
| Fecha de publicación: | 2022 |
| País: | España |
| Recursos: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/329708 |
| Acesso em linha: | http://hdl.handle.net/10261/329708 |
| Access Level: | acceso abierto |
| Palavra-chave: | C3G Rap1 Platelets Ischemia-induced angiogenesis Platelet-mediated metastasis Thrombopoietin c-Mpl RapGEF1 |
| Resumo: | Table S1. Genetically modified animals. Table S2. PCR primers. Table S3. Antibodies used for flow cytometry, confocal immunofluorescence microscopy, immunohistochemistry, immunoprecipitation and western blot. Table S4. Deletion of C3G did not modify platelet counts and its parameters. Platelet number and parameters in C3G-KO mice and their C3G-wt siblings, male and female. The counts were made using an Advia 120 Hematology Analyzer (Bayer). Values are the mean of five, 10-week, mice of each genotype. There were no significant differences between genotypes or genders. Figure S1. Platelet C3G regulates ischemia-induced angiogenesis. (A) 3LL cells were injected in C3G-KO mice and their controls and tumors removed after 15 days. Histograms represent the number of vessels per area (upper) and the vessels surface per area (lower) (mean ± SEM) in tumor sections. (B) Blood collected at the indicated days 5 after implantation of 3LL cells in tgC3G, C3G-KO mice and their controls was incubated with anti-CXCR4-PE and anti-VEGFR-APC to determine the percentage of hemangiocytes. Histograms represent the mean ± SEM of the percentage of CXCR4+ (upper) or VEGFR+ (lower) cells in peripheral blood from the indicated genotypes. (C) Histograms represent the mean ± SEM of the percentage of CXCR4/VEGFR-double positive cells in blood from tgC3G mice and their controls (left) and the percentage of VEGFR-positive cells in blood from C3G-KO mice and their controls (right), collected at the indicated times post-ischemia. (D) Histogram represents the quantification of SDF-1 levels in thrombin-induced secretome from tgC3G, C3G-KO platelets and their controls, using a Mouse Angiogenesis Array Kit (n=2, each per duplicated). Representative images of the arrays are depicted in the left panels. SDF-1 spots are marked with red boxes. a. u, arbitrary units. Reference spots, which are not suitable for quantification, are indicated. (E) Western blot analysis of TSP-1 and VEGF levels in cytosolic (left) or membrane (right) fractions from resting, thrombin (TH)- or ADP- stimulated C3G-KO or C3G-wt platelets. β-actin was used as loading control. Values were normalized to those of resting C3G-wt platelets. Figure S2. C3G ablation or overexpression does not modify physiological MK or platelet production, nor does MK production after TPO injection or 5-FU-induced BM depletion. (A) Box plots showing the median of the number of platelets in C3G-KO mice and their control siblings. (B) Expression of CD41 and CD61 markers in BM cells from the indicated genotypes was determined by flow cytometry using CD41-FITC and CD61-PE. Histograms represent the mean ± SD of the percentage of CD41+, CD61+ or double-positive cells (megakaryocytes). (C) Expression of CD42 (left), CD41 and CD61 markers (middle) and ploidy status (right) was analyzed by flow cytometry in BM from C3G-KO and control mice, 14 days after injection of TPO. (D, E) Expression of CD42 (left), CD41 and CD61 markers (middle) and ploidy status (right) was analyzed by flow cytometry in BM from C3G-KO and C3G-wt mice (D) or tgC3G and wtC3G mice (E), 21 after 5-FU injection. Figure S3. C3G regulates c-Mpl levels and its ubiquitination and interacts with c-Cbl. (A) Western blot analysis of c-Mpl protein levels in platelets (left) or MKs (right) from the indicated genotypes. Values are relative to β-actin expression and were normalized 8 against those of each wild-type. (B) C3G-KO and C3G-wt platelets were treated with thrombin (TH, 0.5 U/ml, 1 min) or TPO (100 ng/ml, 5 min) in the presence or absence or the SFK inhibitor PP2 (10 µM) and labeled with anti-c-Mpl + Alexa FluorTM-568 (red) or anti-Ubiquitin + Alexa FluorTM-647 (green). Upper panels: representative immunofluorescence images of platelets taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SD of the fluorescence intensities of c-Mpl (left) or ubiquitin (right). (C) The graph shows the Pearson’s Correlation Coefficients (mean ± SD) of c-Mpl and ubiquitin under the indicated experimental conditions. (D) tgC3G and wtC3G platelets were treated with TPO (100 ng/ml, 5 min) in the presence or absence of the SFK inhibitor PP2 (10 µM) and labeled with anti-c-Mpl + Alexa FluorTM-568 (red) or anti-Ubiquitin + Alexa FluorTM-647 (green). Left panels: representative immunofluorescence images of platelets of each genotype under each treatment condition, taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SD of the fluorescence intensities of c-Mpl (left) or ubiquitin (right). (E) The graph shows the Pearson’s Correlation Coefficients (mean ± SD) of c-Mpl and ubiquitin under the indicated experimental conditions. (F) TPO induces C3G and c-Cbl colocalization. Representative immunofluorescence images of tgC3G platelets treated with TH (0.5 U/ml, 1 min), ADP (25 µM, 5 min) or TPO (100 ng/ml, 5 min) and labeled with anti-c-Cbl + Alexa FluorTM-568 (red) and anti-C3G + Alexa FluorTM-647 (green). Histograms show the Pearson’s Correlation Coefficients (mean ± SD) of C3G and c-Cbl under the indicated experimental conditions. Figure S4. C3G promotes c-Cbl phosphorylation by Src. (A) Representative immunofluorescence images of tgC3G, C3G-KO and control platelets treated with thrombin (TH, 0.5 U/ml, 1 min) or TPO (100 ng/ml, 5 min) and labeled with antiphospho-c-Cbl + Alexa FluorTM-647 (red) and Phalloidin (green). 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 (p-c-Cbl). (B) Representative immunofluorescence images of C3G-KO platelets and their controls treated with TH (0.5 U/ml, 1 min) or TPO (100 ng/ml, 5 min), in the presence or absence of PP2, 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-Src (pSrc) (upper) and phospho-c-Cbl (lower) under the indicated treatments. (C) Graph showing the Pearson’s Correlation Coefficients (mean ± SD) of phospho-c-Cbl and phospho-Src under the indicated experimental conditions. (D) Representative immunofluorescence images of tgC3G platelets and their controls treated with TPO (100 ng/ml, 5 min), in the presence or absence of PP2, and labeled with anti-phospho-c-Cbl + Alexa Fluor TM-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 ± SEM of the fluorescence intensities of phospho-Src (p-Src) (left) and phospho-c-Cbl (right) under the indicated treatments. |
|---|