Extended Data Fig. 6: p38γ and CDKs cooperate in the induction of Rb phosphorylation and liver proliferation.
From: p38γ is essential for cell cycle progression and liver tumorigenesis
a, In vitro kinase assay, in which phosphorylation sites identified by mass spectrometry are underlined. b, Immunoblot analysis of CDK2 in p38γ immunoprecipitates from the liver of AlbCre-p38γ mice with or without infection with AAV expressing active p38γ (AAVp38γ*). c, Immunoblot analysis of Rb expression in the liver in wild-type and CDK1/2 KO mice (AAV2/8-Cre-infected) in steady state. d, BrdU immunostaining analysis after PHx. Quantification is shown as mean ± s.e.m. n = 5 fields: 0 h, n = 4; 2 h, n = 5; 8 h, n = 5; 12 h, n = 5; 24 h, n = 5; 36 h, n = 7; 48 h, n = 14; 60 h, n = 6; 72 h, n = 4 mice. Comparison was performed using a one-way ANOVA coupled with Bonferroni’s multiple comparison test. e, Immunoblot analysis in livers from AlbCre and AlbCre-p38γ mice. f, Immunoprecipitation–immunoblot analysis of the interaction of CDK2 with wild-type and nonphosphorylatable Rb in HEK-293T cells transfected with human HA-Rb wild-type or HA-Rb ΔCDK (nonphosphorylatable by CDKs). g–m, Wild-type mice were injected with lentivirus containing shScramble control or short hairpin RNA (shRNA) targeting CDK1/2 (shCDK1/2) or CDK4/6 (shCDK4/6) with or without AAV expressing active p38γ (AAVp38γ*). Mice were subjected to PHx or to a sham procedure. g, k, l, Immunoblot analysis. Hepatocyte proliferation was analysed by Ki67 immunostaining 48 h after PHx. Scale bars, 50 μm. h, i, m, Hepatocyte proliferation was analysed 48 h after PHx. h, Ki67 immunostaining. Scale bar, 100 μm. i, Ki67-positive cell quantification is shown as mean ± s.e.m. n = 5–10 counted areas from wild-type mice: 0 h, n = 2; 48 h, n = 3; shCDK1/2 mice: 0 h, n = 3; 48 h, n = 4; shCDK1/2 mice: 0 h, n = 4; 48 h, n = 4; AAVp38γ* mice: 0 h, n = 4; 48 h, n = 5. One-way ANOVA coupled with Bonferroni’s multiple comparison test; ***P < 0.001. m, n = 5 counted areas from n = 5 mice). Scale bar, 100 μm. Comparisons were made by two-sided Student’s t-test; ***P < 0.001. In the western blots, each lane corresponds to a different mouse and is representative of at least three independent experiments.
