Extended Data Fig. 8: Tgfbr2 deletion suppresses plasticity in Atrx^KO organoids.

(a) Representative images of AKP Control and AKP Atrx^KO cells treated or untreated with 5 ng/ml TGF-beta (TGFβ) for 72 h, n = 3 vs 3 independent experiments. Scale bars, 200 μm. (b) Relative cell viability of AKP Control vs AKP Atrx^KO single cells treated or untreated with TGF-beta, n = 3 vs 3 independent experiments. (c) Targeted DNA sequencing of targeted Tgfbr2 locus in AKP Atrx^KO organoid lines. (d) RT-qPCR analysis of representative genes important for squamous, EMT and colonic lineage specification and function in AKP Control, AKP Atrx^KO Control and AKP Atrx^KO Tgfbr2^KO organoids. (e) FACS plots for analysing LY6D^+ve/ITGA5^+ve cells from AKP Atrx^KO Control and AKP Atrx^KO Tgfbr2^KO organoids, n = 3 vs 3 independent experiments. (f) Quantification of percentage of cells in each LY6D / ITGA5 population, n = 3 vs 3 independent experiments. (g) Fluorescence microscopy of phalloidin stained AKP Atrx^KO Control and AKP Atrx^KO Tgfbr2^KO organoids following treatment with 5 ng/ml TGF-beta, n = 3 vs 3 independent experiments. Scale bars, 200 μm. (h) Quantification of presence of spindle -like organoid structures formed following treatment of AKP Atrx^KO Control and AKP Atrx^KO Tgfbr2^KO organoids with 5 ng/ml TGF-beta, n = 3 vs 3 independent experiments. For (b) data are presented as mean values ± SD. P values were calculated using one-sided ANOVA multiple comparisons test. For (d) n = 1 biologically independent sample examined over 3 independent experiments. Gene expression was normalised to Actb and levels relative to AKP Control calculated using the ΔΔCt method. Data are mean ± SD. P values were calculated using one-sided ANOVA multiple comparisons test. For (h) p value was calculated using two-tailed student’s ttest.