Fig. 4.

Cul7 inactivation augments p53 activity. (A) DNA content analysis by flow cytometry after Cul7 knockdown in MCF-7 cells. Cells were treated with 500 ng/ml DOX for 72 h. DNA damage was induced by addition of etoposide (20 μM). MCF-7 cells with and without induction of Cul7-specific or a nonsilencing microRNA were treated with etoposide for the indicated periods. The experiment was repeated twice, and representative results are provided. The % cell cycle distributions are average results of two independent experiments. 2N, G₁ phase; 4N, G₂/M phase. SI Fig. 9B shows a similar analysis 24 h after etoposide treatment. (B) Concomitant knockdown of Cul7 and p53 in MCF-7 cells. MCF-7 EMI-Cul7microRNA cell line was transfected with p53-specific (p53si) or nonsilencing (NonS) siRNAs. Twenty hours later, Cul7 knockdown was induced by addition of DOX for 72 h. Cul7, p53, and β-actin protein levels were analyzed by Western blotting.