The Cyclin B1 and AuroraA protein levels and Cdc25C Ser216 phosphorylation, suggesting that mitosis is partially restored (Fig. 5B). In addition, it reduced the binding of 14-3-3s and CyclinB1/Cdc2 (Fig. 5C). Even so, co-depletion of Cdc7 and MK2 did not avoid HeLa cell death, presumably becauseMK2 depletion alone induced important cell death (information not shown). The time essential for nuclear translocation soon after co-depletion of Cdc7 and MK2 was shortened close for the handle level. Moreover, the G2 elongation observed immediately after Cdc7 depletion was canceled also by co-depletion of ATR or p38 with Cdc7 (Fig. 5D). These results indicate that this G2 checkpoint depends upon ATR-regulated MK2 activation.1-Methylpyrrolidine Cancer Cytoplasmic accumulation of CyclinB1 does not happen in p53-positive U2OS or Aim apoptosis Inhibitors MedChemExpress HCT116 soon after Cdc7 depletionAlthough Cdc7 depletion in U2OS cells (human osteosarcoma), induced vigorous cell death, the levels with the mitotic kinases didPLoS One | plosone.orgCancer Cell Death Induced by Replication DefectFigure 5. MK2 is activated in Cdc7-depleted HeLa cells and is necessary for cytoplasmic accumulation of CyclinB1. (A) HeLa (lanes 1, two, 7 and eight), U2OS (lanes 3 and 4) and NHDF (lanes five and 6) cells had been treated with manage or Cdc7 siRNA and the complete cell extracts have been run on a phosgel and analyzed by western blotting. Lanes 7 and eight, extracts from Cdc7 siRNA-treated HeLa cells have been non-treated (2) or treated with lphosphatase (+). Arrowheads indicate the phosphorylated MK2 band. (B) HeLa cells have been treated with manage siRNA (lanes 1 and five), Cdc7 siRNA (lanes 2 and six), Cdc7 and MK2 siRNAs (lanes three and 7) and MK2 siRNA (lanes four and eight) for 48 hrs, and CSK-soluble (lanes 1; Sup) and -insoluble (lanes 5; Ppt) proteins have been analyzed by western blotting. Tubulin and LaminB are shown as loading controls. (C) Glutathion Sepharose 4B beads carrying GST-14-3-3s protein was incubated for 1 hr at 4uC with CSK-soluble extracts of HeLa cells treated with siRNA, as shown. Bound proteins were examined by Western blotting. “Input” represents only the extracts with no added GST-14-3-3s protein. Cdc7 and MK2 co-depletion decreased the binding between 14-3-3s and Cdc2/CyclinB1. (D) HeLa cells expressing mKO2-CyclinB1 have been treated with indicated siRNAs. The time (hr) involving the first look of cytosolic mKO2-CyclinB1 signal and its translocation in to the nucleus was measured in the time lapse pictures. Co-depletion of MK2, p38 (upstream kinase of MK2) or ATR lowered cytoplasmic retention of CyclinB1 (G2 elongation) was observed in Cdc7-depleted HeLa cells. The P-values of the two-tailed unpaired t-test have been calculated by Prism computer software. Cdc7-D siRNA was used in all of the experiments. doi:10.1371/journal.pone.0036372.gnot raise (Fig. 1). We for that reason established U2OS stably expressing mKO2-CyclinB1, and examined the impact of Cdc7 siRNA around the CyclinB1 dynamics. Within this cell line, we did not observe any accumulation of CyclinB1 inside the cytoplasm just after Cdc7 depletion. The time expected for nuclear translocation in Cdc7depleted U2OS cells was similar to that of control cells (Fig. 6A). Even so, it became longer when p53 was co-depleted (Fig. 6A). The CyclinB1 protein level slightly increased soon after co-depletion of Cdc7 and p53 in U2OS (Fig. 6B). We next examined a colon cancer cell line, HCT116. In p53-positive HCT116 cancer cells, the levels of mitotic proteins like CyclinB1 and Plk1 decreased soon after Cdc7 depletion presumably resulting from G1 arrest (Fig. 7A). Concomitantly, Cdc2/Cycli.