FIG. 3.

RPA32 dephosphorylation at T21/S33 is dispensable for the checkpoint activation but is required for recovery from HU stress. (A) The various cell lines with substitutions in RPA32 (S) were created by stably expressing in HeLa cells the empty vector (V) or Flag-tagged RPA32 variants (WT, VA, and DD) followed by retrovirally silencing endogenous RPA32 in these overexpression cells (O). RPA32 levels in these cell lines were examined by Western blot analysis. (B) WT and DD cells were treated with HU (5 mM). At 0 to 3 h postexposure, cells were fixed and stained with anti-RPA32 antibody. The RPA32 foci were visualized by immunofluorescence, and the percentage of focus-positive cells was determined, normalized, and plotted. Error bars represent the standard deviations from three independent experiments. (C) The RPA32 substitution cells were mock treated (−) or exposed (+) to HU (5 mM), and phosphorylation of Chk1 at S345 and Rad17 at S645 was examined by Western blotting 3 h later. (D) Various cells were irradiated with 25 J/m² UV, and 3 h later, the DNA synthesis rate was determined and normalized to that in untreated cells (Untr). (E) The RPA32 substitution cells were left untreated or irradiated with 25 J/m² UV. Later (1.5 h later), the cells were costained with propidium iodide and anti-phospho-H3 antibodies, and the mitotic fractions were determined by fluorescence-activated cell sorting analysis and normalized to that of unperturbed cells. (F) The RPA32 substitution cell lines were pulse exposed to 0 to 1.2 mM HU for 24 h and then allowed to recover in drug-free medium for 10 to 14 days. The cell viability of various lineages was analyzed by the clonogenic survival assay. All the data points in panels D, E, and F represent the means ± standard deviations (error bars) from three independent experiments, each performed in triplicate.