Extended Data Figure 3: Identification of Yap regulated genes.
From: Yap-dependent reprogramming of Lgr5+ stem cells drives intestinal regeneration and cancer
a, Schematic representation of YapTg mice. Induction of HA–Yap was achieved by intercrossing villin-cre or villin-creERT2 mice with Rosa26-lox-STOP-lox-rtta-IRES-EGFP mice (see Methods). b, Analysis of HA–Yap protein expression in the intestinal epithelium. Intestinal crypts were isolated, lysed and subjected to SDS–PAGE (left panel). Expression of HA–Yap is only detected in transgenic mice in the presence doxycycline. Immunohistochemistry staining using anti-HA and Ki67 antibodies in untreated small intestine of YapTg mice (right panel). c, Analysis of Yap overexpression in organoid cultures. Crypts from YapTg intestines were seeded and induced with doxycycline. Representative organoids cultured for 3 days are shown as bright-field images or Edu (red) and caspase 3 (yellow) stainings. Yap transgene expression was detected by anti-HA staining (green) (n = 5). Arrowhead indicates diminished Edu incorporation in doxycycline-induced organoids. Scale bars, 70 μm. d, Identification of relative expression of Yap-regulated genes by RNA-seq analysis are shown as rank order plots comparing control and Yap+/Δ and YapΔ/Δ organoids isolated at day 1, as well as doxycycline treated and untreated YapTg organoids at day 1 of culture: combined fold change = log2 [(YapΔ/Δ/Yap+/Δ)/YapTg(Dox+/Dox–)]. e, f, qPCR analysis of selected Yap-regulated genes comparing fold change between Yap+/Δ and YapΔ/Δ or doxycycline-treated and untreated YapTg organoids at day 1, respectively. Error bars indicate s.e.m.; n> 3 (n represents the number of independent organoid cultures per genotype per mouse analysed for each gene).
