Figure 2. Brg1 loss attenuates Wnt target gene expression and prevents mislocalisation of Paneth cells and formation of aberrant organoids.

A. Scoring of the lysozyme positive cells revealed a significant increase in number of Paneth cells in VillinCreER^T2+Apc^fl/fl (grey bar) mice compared to double knock-out (black bar) and control (white bar) mice. No difference in Paneth cell numbers was observed between double knock-out and control epithelium. Data are shown as mean ± pooled standard deviation, p value was calculated by means of one way ANOVA, n = 4. B. Analysis of cumulative frequency of Paneth cells at each position along crypt-villus axis revealed their expansion into the upper portion of the crypt in VillinCreER^T2+Apc^fl/fl epithelium compared to double knock-out and control mice (Kolmogorov-Smirnov test p<0.001, n = 4). No difference in Paneth cell distribution was observed between double knock-out and control epithelium (Kolmogorov-Smirnov test p = 0.17, n = 4). C. Organoid culture of crypts derived from VillinCreER^T2+Apc^fl/fl epithelium produced large cystic organoids by day 6 of culture (day 9 post induction). Meanwhile, very few large cysts developed from VillinCreER^T2+Apc^fl/flBrg^fl/fl crypts, with most crypts producing compact simple organoids. Crypts from control intestinal epithelium developed into complex organoid structures by the same time point. Scale bars represent 200 µm. D. Western-blotting analysis of total protein pooled from 3 different animals for each genotype did not reveal changes in the levels of activated β-catenin between VillinCreER^T2+Apc^fl/fl and VillinCreER^T2+Apc^fl/flBrg^fl/fl epithelial samples. Substantially weaker signal was detected in the samples from VillinCreER^T2− control animals. E. qRT-PCR analysis of total RNA extracted from the small intestinal epithelium revealed significantly increased expression of β-catenin and a range of Wnt target genes in VillinCreER^T2+Apc^fl/fl (grey bars) and VillinCreER^T2+Apc^fl/flBrg^fl/fl (black bars) samples compared to control VillinCreER^T2− (white bars) animals. Expression levels of c-Myc, CD44, CyclinD1 and Ascl2, but not β-catenin or Axin2 genes were found to be significantly lower in double knock-out epithelium compared to VillinCreER^T2+Apc^fl/fl samples. Mann-Whitney U-test was performed on ΔΔCt values, * - p<0.05, n≥4. F. Clustering analysis of genes differentially expressed between VillinCreER^T2+Apc^fl/fl and VillinCreER^T2− control epithelium revealed that the same genes in VillinCreER^T2+Apc^fl/flBrg^fl/fl epithelium largely assumed intermediate expression values between those of the other two groups. G. Venn diagram demonstrating overlap between pairwise comparisons of differentially expressed gene signatures within various cohorts.