Extended Data Fig. 9: Fibroblast-specific depletion of CCM3 induces tissue remodelling and spontaneous metastasis.
Validation of stable knockdown of CCM3 using CRISPR/Cas9 technology in V-CAF. a, Immunofluorescence validation of YAP/TAZ translocation to the nucleus of CRISPR/Cas9-modified V-CAFs. Scale bar, 50 μm. b, Immunoblotting analysis of CCM3-knockdown efficiency, α-SMA expression, and activation of pY397-FAK and pS19-MLC2. c, Growth curve of orthotopic 4T1 mammary tumours in nude mice. n = 10 mice from two independent animal experiments. Line and error bars indicate mean ± s.d.; one-way ANOVA. d, Picrosirius red (collagen I and III) staining of paraffin-embedded primary tumour sections. Scatter plots show quantification of percentage of cell area covered by fibrillar collagen. Each data point represents an individual tumour from two independent animal experiments. n = 10 individual tumours. Line and error bars indicate mean ± s.d.; one-way ANOVA. e-f, Immunofluorescence analysis of primary tumour sections stained for αSMA and pS19-MLC2 (all green), DAPI (blue), and some cases for human-specific vimentin (red). Scatter plots show quantification of percentage of cell area covered by αSMA and pS19-MLC2. Each data point represents an individual tumour from two independent animal experiments. n = 10 individual tumours. Line and error bars indicate mean ± s.d.; one-way ANOVA. Scale bars, 50 μm. g, IHC staining of paraffin-embedded primary tumour sections. The sections stained for YAP show nuclear localization of YAP in CCM3-depleted V-CAFs. n = 10 mice from two independent animal experiments. Scale bar, 50 μm. h-i, H&E staining of paraffin-embedded tumour sections of (h) lung and (i) liver metastases (see arrows). j, Scatter plots show the quantification of spontaneous metastases. Each data point represents individual mice quantified from two independent animal experiments. n = 10 mice. Line and error bars indicate mean ± s.d.; one-way ANOVA. Scale bars, 100 μm. Statistical data and uncropped blots are provided in the source data.
