Analysis of p53 and DDB2 functionality and in melanoma cells and melanocytes
(A) Western blot analyses were performed with extracts from the indicated cell lines harvested at various times post UV-irradiation (12.5 J/m2) to monitor p53 functionality, as determined by induction of p21 and DDB2 protein expression. (B) Based on p53 functionality determined in (A), CPD repair measurements for the individual cell lines (Figure 3) were pooled and re-analyzed as a function of p53 status. Data show the average (and standard deviation) of CPD repair for the seven p53 mutant cell lines (RPMI was excluded) and four p53 wild-type cell lines. Asterisks indicate a statistically significant difference (p<0.01; two-tailed Student's t-test) in CPD repair between p53 mutant and wild-type cell lines. (C) Knockdown of p53 in SK-Mel-103 cells inhibits CPD repair. SK-Mel-103 (p53 wild-type) and SK-Mel-187 (p53 mutant) cells were transfected with non-targeting or p53 siRNAs, exposed to UV, and CPD repair measured at the indicated time points. (D) Electrophoretic mobility shift assay of UV-DDB binding to damaged DNA. Cell-free extract from SK-Mel-103 (p53 wild-type) and SK-Mel-187 (p53 mutant) cells were incubated with radiolabeled [6-4] PP substrate DNA and complexes separated on a 5% non-denaturing gel. Purified UV-DDB complex was used as a positive control and CHO-AA8 extract as a negative control. The cell-free extract protein concentrations ranged from 0.5, 1.0, 2.0 or 4.0 μgs per reaction. The experiment was repeated three times, and the average percent binding is indicated.