Extended Data Fig. 5: Effect of ARID1A depletion in hepatocytes on tumour formation.

a, qPCR analysis of Nf2 and Arid1a expression in the livers of control (n = 6 mice), and Nf2 (n = 6 mice), Arid1a (n = 7 mice) and Nf2/Arid1a (n = 7 mice) liver mutant (LKO) mice, four months after tamoxifen treatment. All animals were included. Mean and data for individual mice are shown. b, Livers of control (Arid1a^fl/fl) and Arid1a LKO (Alb^creERT2Arid1a^fl/fl) mice were collected two weeks after tamoxifen treatment, and genomic DNA and proteins were extracted using standard procedures. Representative results are shown, experiments were repeated on four mice for each genotype. Left, PCR analysis of the indicated alleles. Right, western blots of GAPDH (loading control) and ARID1A. c, YAP immunohistochemistry (IHC) staining in control and Nf2 mutant livers. Scale bars, 40 μm. Representative images of experiments that were independently replicated using three mice for each genotype, with similar results. d, Continuation of Fig. 2e. Representative cytokeratin (CK; top) and Ki-67 (bottom) stainings of sections of livers of the indicated genotypes (same genotypes as in Fig. 1d, e and Extended Data Fig. 5a). Note intrahepatic cholangiocarcinomas (iCCA; CK⁺Ki-67⁺) and hepatocellular carcinomas (HCC; Ki-67⁺CK⁻) were found only in livers from Nf2/Arid1a LKO mice. Scale bars, 100 μm. Representative images are shown, experiments independently replicated for all of the mice of each genotype described in a, with similar results. e, qPCR analysis of selected genes of livers of mice with the indicated genotypes. All animals were included. Data are normalized to Nf2/Arid1a LKO mice. Data are mean + s.d. for same number of mice per genotype as in a. f, Continuation of Fig. 2f. Control, Arid1a LKO and Arid1a/Yap/Taz LKO mice were treated with tamoxifen and were then fed a DDC-containing diet for six weeks. CK (top; scale bars, 40 μm) and Ki-67 (bottom; scale bars, 20 μm) stainings of liver sections from the indicated mice. Note the presence of early cholangiocarcinoma lesions (CK⁺Ki-67⁺) in the Arid1a LKO mice and their absence upon concomitant YAP/TAZ loss (that is, in the Arid1a/Yap/Taz LKO mice). Asterisks indicate porfirin deposits, which are typically present in the liver of mice treated with DDC. Representative images are shown, experiments were independently replicated for all of the mice of each genotype (same number of mice as in Fig. 2f), with similar results. g, Representative qPCR analysis of Afp expression in the livers of control (n = 4), Arid1a LKO (n = 5), Arid1a/Yap/Taz LKO (n = 5) mice treated with tamoxifen and then DDC. Data are normalized to livers of mice not treated with DDC (n = 4). Data are mean + s.d. of the indicated number of mice. This experiment was independently repeated three times with similar results, analysing, in total, at least 10 mice for each genotype. h, Representative E-cadherin staining showing that CCA lesions retain an epithelial morphology in sections of the liver of the indicated genotype. Scale bar, 30 μm. Experiments were independently repeated on three DDC-treated Arid1a LKO mice, with similar results. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test (a) or with Tukey’s multiple comparisons test (e, g).

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