Figure 4.

ATR Activation at the NE Does Not Depend on RPA

(A) Confocal images of HeLa cells exposed to normal or hypertonic medium (0.5 M sorbitol; 20 min). Samples were stained with anti-ATR (green), -RPA32 (red) Abs, and DAPI.

(B) HeLa cells were transfected with empty vector or RPA70 shRNA and selected with 3 μg/ml puromycin (72 hr). Cells were incubated with hypertonic medium containing 0.5 M sorbitol for 20 min and stained with anti-ATR (green), -RPA70 (magenta) Abs, S-phase-specific EdU (red), and DAPI (blue). RPA70 protein levels were analyzed by western blot using anti-RPA70 Ab and anti-tubulin as a loading control (lower panel). The graph shows the percent ATR fluorescence intensity in different cellular compartments on a set of cells (n = 20).

(C) Puromycin-selected RPA70 shRNA-transfected HeLa cells were incubated with hypertonic medium containing 0.5 M sorbitol for 30 min and stained with p-Chk1 (green) and DAPI (blue). Graphs show the integrated p-Chk1 fluorescence intensity on a set of cells (n = 20). Quantitative analysis of P-Chk1 levels is shown.

See also Figure S4.