Figure 4

Viability of Baf53a-deficient ES cells is repressed. (A) Baf53a cKO ES cells were cultured for 2–6 days in 1 μg/mL Tet and their morphologies were examined on the days indicated. Scale bar is 250 μm. (B) Baf53a cKO ES cells were seeded at 1000 cells per well in a 96-well dish, cultured with or without 1 μg/mL Tet, and subjected to the WST-1 assay after 4 days. The data represents three independent experiments and the means ± standard deviations are shown. **p < 0.01 (t-test). (C) Growth curves of Baf53a cKO ES cells. Baf53a cKO ES cells were cultured in the presence or absence of 1 μg/mL Tet and the number of cells was counted on the days indicated. The result is the means ± standard deviations of three independent experiments. (D) Baf53a cKO ES cells were cultured with or without 1 μg/mL Tet for 2–4 days, and protein expression of Baf53a, p53, and cleaved Caspase 3 was examined by Western blot analysis. αTubulin was used as a loading control. The data is representative of three independent experiments (left panel). Signal intensity of bands of cleaved Caspase 3 was measured by a densitometry and the result is the means ± standard deviations of three independent experiments (right panel). *p < 0.05 (t-test). (E) Expression of p21 mRNA was examined by quantitative RT-PCR. The data was normalized to GAPDH expression, and the result is the means ± standard deviations of three independent experiments. *p < 0.05 (t-test). (F) Baf53a cKO ES cells were cultured with or without 1 μg/mL Tet for 2–4 days, and protein expression of Baf53a and PCNA was examined by Western blot analysis. αTubulin was used as a loading control. The data is representative of two independent experiments. (G) Baf53a cKO ES cells were cultured with or without 1 μg/mL Tet for 4 days, and protein expression of Ki67 was examined by immunofluorescent stain. Nuclei were stained with DAPI. Scale bar is 50 μm. The data is representative of two independent experiments.