Extended Data Fig. 8: Activation of FAK–YAP signalling after chemotherapy.
From: Cell–matrix interface regulates dormancy in human colon cancer stem cells
a. Gene set enrichment analysis of COL17A1-overexpressed (OE) versus KO CCO32 organoids using YAP target genes. The YAP target genes derive from ChIP-seq-validated YAP/TAZ targets in Zanconato F., et al43. NES; normalized enrichment score. p = 8.3 x 10–14. b. Gene set enrichment analysis of LGR5+p27+ versus LGR5+p27– cells sorted from CCO7, CCO12, CCO20 and CCO32 using the same YAP target genes. p = 1.1 x 10–42, FDR = 6.1 x 10–42. c. Relative expression of YAP/TAZ/TEAD target genes (LATS2, CTGF and CYR61) (mean ± s.e.m.) in TEADi-treated CCO32 organoids compared to the control. The organoids were cultured with or without a TEADi (MYF-01-37, 20μM) for 2 days. Statistics, two-sided Welch’s t-test. The exact p-value for LATS2 is 2.1 x 10–6. d. The expression of the COL17A1 protein in COL17A1-OE CCO7 and CCO32 organoids after treatment with CPT (50 nM for CCO7, 10 nM for CCO32). COL17A1-rescued, COL17A1 KO organoids (COL17A1 KO + OE) were used. COL17A1 was induced by a 2-day treatment with 500 ng/ml Dox. Organoid lysates were collected 6, 24 and 48 hr after a treatment with CPT. The figures on the bottom of the images show the protein areas analysed by the Compass software. The values were scaled to the Dox+ condition. e. The expression of COL17A1, phospho-FAK (pFAK) and total FAK (tFAK) protein in CCO7 and CCO32 organoids after treatment with 5-FU (30 μM) or oxaliplatin (30 μM for CCO32, 50 μM for CCO7). Organoid lysates were collected 6, 24 and 48 hr after a treatment with 5-FU or oxaliplatin. f. The expression of MMP genes in CPT-treated CCO32 organoids. MMP genes with at least a transcript count > 1 in more than a half of the samples are shown. g. Capillary-based immunoassays of COL17A1 using CCO32 organoids with or without CPT treatment (10 nM). CPT-treated organoids were treated with vehicle or two different MMP inhibitor (llomastat (10 μM) or Marimastat (3 μM)). Organoid lysates were collected 48 h after treatment. h. Whole-mount immunostaining of phosho-FAK (green) in CCO7 organoids with or without CPT (50 nM, 48 hr) and FAKi (PF573228, 10 μM, 48 hr) treatments. i. The ratio of phosho-FAK-positive area to nuclei area (mean ± s.e.m.) in CCO7 organoids with or without the indicated treatments. Each dot shows one organoid. Statistics, two-sided Welch’s t-test. j. Whole-mount immunostaining of YAP (magenta) in CCO32 organoids with or without CPT (10 nM, 48 hr) and FAKi (10 μM, 48 hr) treatments. k. The proportion of nuclear YAP-positive cells (mean ± s.e.m.) in CCO7 and CCO32 organoids with or without CPT (50 nM, 48 hr for CCO7 and 10 nM, 48 hr for CCO32) and FAKi (10 μM, 48 hr) treatments. Each dot represents one organoid. From the left, p = 1.5 x 10−6, p = 3.9 x 10–9, p = 9.1 x 10–11, two-sided Welch’s t-test. l. Representative confocal images of YAP staining (magenta, left) with p27-mVenus (p27mV) reporter (green, left) and phospho-FAK (green, right) expressions in COL17A1-KO or OE CCO7 with or without CPT treatment (50 nM, 48 hr). COL17A1 was induced by a 2-day pre-treatment with 500 ng/ml Dox. m. The proportion of nuclear YAP- or pFAK positive cells per nuclei (mean ± s.e.m.) in COL17A1-KO or OE CCO7 organoids with or without the indicated treatments. Each dot shows one organoid. Statistics, two-sided Welch’s t-test. Scale bars: 25 μm (l), 50 μm (h, j)
