Extended Data Fig. 7: Characterization of V-CAFs stably expressing GFP, CCM3–GFP, CCM3-mutant-GFP or FAT–GFP.
a, Immunoblotting against GFP. b, Gel contraction of the four V-CAF lines. n = 4 independent experiments. Line and error bars indicate mean ± s.d; one-way ANOVA test, following up multiple comparisons. c, Immunoblotting analyses against αSMA, pS109-YAP, pS19-MLC2 and tubulin. d, Representative images of the GFP-tagged constructs (green) and YAP/TAZ (red) staining’s. Quantification of YAP/TAZ nuclear/cytoplasmic ratio. Each data point represents an individual cells. n = 30 cells. Line and error bars indicate mean ± s.d.; one-way ANOVA test, following up multiple comparisons. ns: non-significant. Scale bars, 20 μm. e-f, Knockdown efficiency of endogenous CCM3 (e) mRNA and (f) protein levels using siRNAs targeting the UTR. Line and error bars indicate mean ± s.d. Notice, that the exogenous CCM3–GFP is not depleted by the siRNAs. g, Immunofluorescence analysis of YAP/TAZ in V-CAFs depleted with siRNAs targeting CCM3 UTR region and stained for YAP/TAZ (green). Quantification of YAP/TAZ nuclear/cytoplasmic ratio. Each data point represents an individual cells. n = 20 cells. Line and error bars indicate mean ± s.d.; one-way ANOVA test, following up multiple comparisons. Scale bars, 50 μm. h, Flow cytometry analysis shows that overexpression of CCM3–GFP decreases the levels of pY397-FAK and pY118-paxillin in V-CAFs and Cer-CAFs. Statistical data and uncropped blots are provided in the source data.
