Extended Data Fig. 2: CCM3 is a negative regulator of YAP/TAZ activation.
a, Immunofluorescence analysis of YAP/TAZ in V-CAFs depleted with three different siRNAs targeting CCM3 and stained for YAP/TAZ (green). Scale bars, 50 μm. b, Quantitative PCR (qRT-PCR) of selected YAP/TAZ target genes. Bars show mean ± s.d. mRNA levels normalized to tubulin levels after 72 h of transfection. n = 4 independent experiments; one-way ANOVA test. c, Images show fibroblast-induced contraction after 72 h of remodelling. The scatter plots show quantification of fibroblast-induced contraction relative to siRNA control (siCtr) cells. Each data point represents an independent experiment. n: independent experiments (Mouse mammary NF: 3; Mouse mammary CAF: 3; Human lung NF: 4; Human lung CAF: 5; Human CD26high NF: 3). Line and error bars indicate mean ± s.d.; one-way ANOVA test. d, Immunoblotting analyses of siCCM3-depleted murine normal fibroblasts (NF). e, Immunofluorescence analysis of YAP/TAZ (green) after loss of CCM3 in murine fibroblasts isolated from the PyMT-MMTV mouse model at different progression stages. siRNA-depletion of CCM3 induces nuclear YAP/TAZ localization in four isolates of normal fibroblasts (NF1-4), one isolate of hyperplasia-associated fibroblasts (HpAF), one isolate of adenocarcinoma-associated fibroblasts (AdAF), and four isolates of carcinoma-associated fibroblasts (mCAF1-4). Scale bars, 50 μm. f, Immunofluorescence analysis of YAP/TAZ (green) after loss of CCM3 in matched CAFs and normal lung fibroblasts isolated from a patient with non-small cell lung carcinomas and from human interlobular breast fibroblasts (CD105low / CD26high) isolated from healthy patients. g, Quantification of the YAP/TAZ nuclear/cytoplasmic ratio in fibroblastic isolates. Each data point represents individual cells from three independent experiments. n: individual cells (Mouse mammary NF, 50. Mouse mammary CAF: siCtr, 49; siCCM3, 55. Human lung NF, 60. Human lung CAF, 60. Human CD26high NF, 60.). Line and error bars indicate mean ± s.d.; one-way ANOVA test. Statistical data and uncropped blots are provided in the source data.
