Extended Data Fig. 6: p38γ and CDKs cooperate in the induction of Rb phosphorylation and liver proliferation. | Nature

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

Extended Data Fig. 6

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.

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