Fig. 4. (A) Downregulation of Grp/DChk1 protein levels in S2 cells using RNAi. Grp/DChk1 expression in S2 cells (Fig. 4A, lane 1) and S2 cells treated with dsRNA of Grp/DChk1 (Fig. 4A, lane 2). Incubation of S2 cells with dsRNA of Grp/DChk1 for 96 hours reduced Grp/Dchk1 expression to below detection levels. As a loading control, protein levels of -tubulin were detected. (B) Grp/DChk1-depleted cells are defective in accomplishing cell-cycle arrest in response to HU or IR. Grp/DChk1 levels were downregulated by RNAi before cells were treated with 10 mM HU for 15 hours or treated with 150 Gy ionizing radiation followed by 2 hours of recovery. Grp/DChk1-depleted cells positively stained for PH3 were counted using flow cytometry. In Grp/DChk1-depleted cells left untreated 5.1% of the cells was in mitosis. In Grp/DChk1-depleted cells treated with HU also 5.1% of the cells was in mitosis and, in IR-treated Grp/DChk1-depleted cells, 4.4% of the cells were in mitosis. Error bars represent s.e.m. (n=13).