Accumulation of the cells in the S-phase is not sufficient for large nucleolar TOPBP1 foci formation. (A) Image-based analysis of cell-cycle distribution of control HeLa cells and cells treated with APH (1 µM, 12 h) or HU (1 mM, 12 h). (B) HeLa cells were synchronized in the S phase by treatment with nocodazole (NOC) for 12 h, incubation in fresh medium for an additional 9 h, and then treatment with APH (1 µM) for different periods (1, 3, 6, and 12 h). Synchronization accuracy was monitored using image-based analysis of cell-cycle distribution shown in the middle. Control represents the cells that were NOC synchronized in the S phase but not treated with APH. The cells were immunostained for TOPBP1. Percentage of cells containing large TOPBP1 foci is shown. (C) Representative image of HeLa cells that were treated with APH (1 µM, 12 h) and immunostained for TOPBP1 (green) and ETAA1 (magenta). The DNA was stained with DAPI (gray). neg., negative; pos., positive.