Figure 4.

Genetic ablation of p21^Cip, p16^Ink4a or p19^Arf does not allow proliferation of DKO MEFs. (A) Western blot analysis of protein lysates of indicated MEFs expressing either control shRNA (C) or Hdac1 shRNA (KD) for Hdac1, Hdac2 and p21^Cip. Cdk4 served as a loading control. (B) Representative pictures of MEFs with indicated genotypes infected with retroviruses expressing either control shRNA or Hdac1 shRNA. (C) Growth curve analysis of Hdac2KO;p21^+/+(squares), Hdac2WT;p21^−/− (triangles), Hdac2KO;p21^+/− (diamonds) and Hdac2KO;p21^−/− (circles), MEFs expressing either control shRNA (left panel) or Hdac1 shRNA (right panel). (D) Western blot analysis of protein lysates for Hdac1 and Hdac2 of two independent Hdac2WT;p21^−/− and Hdac2KO;p21^−/− MEF clones infected with either control (#1, #3, #5, #7) or Hdac1 shRNA (#2, #4, #6, #8), isolated at day 8 of the growth curve analysis as shown in (C). The clone numbers in (C) correspond with the clones and genotypes as shown in (D). Cdk4 served as a loading control. (E) Left panel: western blot analysis of Hdac2WT;Cdkn2a^−/− and Hdac2KO;Cdkn2a^−/− MEFs expressing either control (control) or Hdac1 shRNA (Hdac1KD) for Hdac1, Hdac2, p16^Ink4a and p19^Arf. Cdk4 was used as a loading control. As a control for p16^Ink4a and p19^Arf expression, we used late passage wild-type MEFs. Right panel: growth curve analysis of Hdac2WT;Cdkn2a^−/− (squares) and Hdac2KO;Cdkn2a^−/− (circles) MEFs expressing either control (filled tags) or Hdac1 shRNA (open tags).