Figure 8.

Hsf1 knockdown in SK-N-SH cells suppresses p53 nuclear localization. (A) SK-N-SH cells were grown to 60% confluence and then transfected with the indicated siRNA either once (1x) or twice with a 24-hour interval (2x). Forty-eight hours after the last transfection of siRNA, the transfected cells were either treated with a 5-Gy ionizing radiation or left untreated. Cells were harvested, and extracts were prepared 3 hours after irradiation. Hsf1 expression was determined by Western blot analysis using an anti-Hsf1 antibody. β-Actin levels are depicted as the loading control. (B) SK-N-SH cells were grown on coverslips at 37°C to 60% confluence and were transfected with the indicated siRNA. The cells were transfected twice with the Hsf1 siRNA as in panel (A). Forty-eight hours later, the cells were either treated with a 5-Gy ionizing radiation or left untreated. p53 localization was stained using DO1 antibody 3 hours after irradiation. (C) The fraction of cells with nuclear localized p53 in panel (B) was quantified. Graphs show the mean ± SE from three independent experiments.