Figure 4

p53 mediated growth arrest of G-1 in ER− breast cancer cells. SkBr3 cells were treated with 1 μM G-1 for the indicated time periods, and then mRNA levels of p53 (a) and MDM2 (b) were quantified by real-time PCR, the protein levels of p53 (c) were detected by western blotting. SkBr3 cells transfected with si-p53 or si-NC for 24 h, and the mRNA and protein expression of p-53 were measured by qRT-PCR and western blotting (d), respectively. The transfected cells were then stimulated with or without G-1 (1 μM) for another 24 h, the cell viability was assessed by CCK-8 kit (e). (f) After treatment with G-1 for 24 h, nuclear and cytoplasmic cellular fractions were isolated by differential lysis. The levels of p53 in nuclear and cytoplasmic cellular fractions were detected by western blotting. (g) SkBr3 cells were treated with or without G-1 (1 μM) for 24 h. After fixation, the cellular location of p53 (red) was examined by immunofluorescence staining and nuclei were stained with Hoechst (blue). (h) SkBr3 cells treated with 1 μM G-1 for the indicated time periods. After p53 was immunoprecipitated from equal amount of lysates, the ubiquitination of p53 was examined by Western blotting. (i) SkBr3 cells were treated with 1 μM G-1 for the indicated time periods, and then p-Ser¹⁵-p53 and p53 were measured by western blotting. Data were presented as means±S.D. of three independent experiments. *P<0.05 compared with control