Fig. 3

Effect of miR-200KO and Zeb1²⁰⁰ mutations on Zeb1 and downstream target regulation. a Mean log₂FC (with 95% confidence intervals) of Cdh1 and Zeb1 in islets. b Immunoblots evaluating ZEB1 and CDH1 in islets (n = 2 mice per lane). Quantification is normalized to β-actin and relative to RT2. c Allelic expression of mutant Zeb1 allele in three RT2_Zeb1^200H mice. d ZEB1 quantification in end-stage tumors (immunoblots in Supplementary Figure 3b) (n = 10−13 tumors), normalized to β-actin and calibrator. e, f Cumulative distributions of log₂FC of Zeb1 targets in islets of e RT2_DKO vs. RT2 and f RT2_Zeb1^200M vs. RT2. g Hierarchical clustering and heat-map analysis of Zeb1 targets in islets (scale bar represents Euclidian distance between samples). h, i Cumulative distributions of log₂FC of miR-200c predicted targets subdivided by context+ score in islets of h RT2_DKO vs. RT2 and i RT2_Zeb1^200M vs. RT2 mice. P values shown are for context+ <–0.3. j Percent survival of RT2 mice with WT or mutant Zeb1 3′UTR and endogenous or overexpressed miR-200 levels (RT2, RT2_RipmiR-141~200c, RT2_Zeb1^200H_RipmiR-141~200c, RT2_Zeb1^200M_RipmiR-141~200c, RT2_Zeb1^200H, RT2_Zeb1^200M, n = 42, 8, 13, 8, 45, 26, respectively). k Blood glucose (RT2, RT2_RipmiR-141~200c, RT2_Zeb1^200H_RipmiR-141~200c, RT2_Zeb1^200M_RipmiR-141~200c, RT2_Zeb1^200H, RT2_Zeb1^200M, n = 11, 8, 9, 9, 11, 10, respectively). Dotted line represents onset of severe hypoglycemia. l Percent tumors per grade (RT2, RT2_Zeb1^200M, RT2_Zeb1^200H_RipmiR-141~200c, RT2_Zeb1^200M_RipmiR-141~200c, from n = 5, 5, 8, 5 mice and n = 55, 16, 24, 15 tumors, respectively). m Percent insulin-positive and -negative tumors (RT2, RT2_Zeb1^200M, RT2_Zeb1^200H_RipmiR-141~200c, RT2_Zeb1^200M_RipmiR-141~200c, from n = 5, 5, 8, 5 mice and n = 67, 15, 24, 15 tumors, respectively). n Percent animals with insulin-positive and -negative metastases (RT2_Zeb1^200H, RT2_Zeb1^200M, RT2_Zeb1^200H_RipmiR-141~200c, RT2_Zeb1^200M_RipmiR-141~200c, n = 7, 9, 8, 5, respectively). o Representative insulin and SV40 immunofluorescence of RT2_Zeb1^200M_RipmiR-141~200c liver metastases (scale bar = 50 μm). a, c, e–i RNA sequencing of islets of 6-week-old mice (RT2, RT2_DKO, RT2_Zeb1^200H, RT2_Zeb1^200M, n = 2, 5, 3, 4, respectively). d, k Data are plotted as mean ± SD. Significance was evaluated by a empirical Bayes method, d one-way ANOVA with Dunnett’s multiple comparisons (vs. RT2), e, f, h, i competitive gene set test, j Mantel Cox test, and k two-tailed t test with Holm−Sidak correction (vs. RT2). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001