Figure 6.
ARID1B controls senescence by coordinated regulation of multiple targets. (A) NrasG12V or NrasG12V/D38A transposon constructs were delivered into mouse livers by hydrodynamic injection. Livers were harvested after 6 d, followed by mRNA extraction and quantitative RT–PCR analysis for Entpd7, Slc31a2, and Ndst2 mRNA levels. n = 6 mice per condition were used. (B) Transposon constructs carrying NrasG12V cDNA and Arid1b shRNAs (shSL.1 and shSL.2) or control (shCTR) were delivered into mouse livers by hydrodynamic injection. n = 4 mice per condition were used. Livers were harvested after 6 d, and sections were stained for Nras and Ki67. Quantification of IF staining is shown at the right. Graphs represent mean ± SD. (***) P < 0.001; (**) P < 0.005; (*) P < 0.05; (n.s.) nonsignificant by two-tailed Student t-test. (C,D) Transposon constructs carrying NrasG12V cDNA and combinations of two control shRNAs, one control and one targeting Arid1b (shAR.1), one control and one targeting Entpd7 (shEN.1), one control and one targeting Slc31a2 (shSL.2), and a combination of Entpd7 and Slc31a2 shRNAs (shEN.1 + shSL.2) were delivered into mouse livers by hydrodynamic injection. In this experiment, a more active transposase (SB100) that allows integration of multiple shRNAs was used. n = 3 mice per condition were used. Livers were harvested after 9 d, and sections were stained for Nras, Ki67, and SA-β-Gal. Representative pictures of IHC for Nras and SA-β-Gal staining are shown. Quantification of the staining is shown at the bottom. Bar, 100 μm. Graphs represents mean ± SD. (***) P < 0.001; (**) P < 0.005; (*) P < 0.05; (n.s.) nonsignificant by two-tailed Student t-test.