Figure 4.
Knockdown of RNF20 compromises p53 activation. (A) MCF10A cells were transiently transfected with RNF20 siRNA (siRNF20) or LacZ siRNA (siLacZ) as control. Forty-eight hours later, cells were treated with NCS (200 ng/μL) for 2 and 5 h, and harvested. Equal amounts of protein from each sample were resolved by SDS-PAGE, followed by Western blot analysis with antibodies against p53 (DO1+PAb1801), RNF20, p21, H2Bub, and GAPDH. (B) HCT116 cells were transiently transfected with RNF20 siRNA, p53 siRNA, or LacZ siRNA as control. Forty-eight hours later cells were treated with 5-FU (50 μg/mL) for 24 h, harvested, fixed, and subjected to FACS-assisted DNA content analysis. (NT) Nontreated. (C) Quantification of the percentage of cells with sub-G1 DNA content, indicative of apoptosis, in HCT116 cells treated or not treated with 5-FU. Values were averaged from three independent experiments as in B. (**) P-value <0.01. (D) U2OS cells were transfected as in B. Forty-eight hours later, cells were treated with NCS (200 ng/μL) for 8 or 16 h, and BrdU incorporation was analyzed by FACS. The Y-axis indicates the percentage of BrdU-positive cells. (NT) Nontreated. Numerical values above each bar denote the extent of BrdU incorporation relative to the nontreated sample of the corresponding siRNA-transfected population.