Fig. 6

Suppression of the S phase checkpoint upon CRL4 depletion. a HeLa cells were siRNA-transfected and incubated for 24 h with cisplatin. The resulting cell cycle distribution was analyzed by flow cytometry. Rectangles contain S phase cells. b Quantification of EdU incorporation in S phase cells after the indicated treatments. EdU intensities are normalized to siNC controls not exposed to cisplatin (N = 5). c Immunoblot showing pCHK1 (phosphorylated at Ser345) after exposure to 20 μM cisplatin. d Quantification of pCHK1 normalized to GAPDH. Values are expressed relative to the amount of pCHK1 in CRL4-proficient cells treated for 24 h with cisplatin (N = 3–5). e CUL4A/B-depleted cells were incubated for 24 h with cisplatin as indicated. Mitotic cells were stained with antibodies against pH3 and DAPI, and analyzed by flow cytometry. Rectangles indicate pH3-positive cells. f Percentage of mitotic cells resulting from the indicated treatments (N = 3–5). In panels b, d, f, data are presented as mean ± SEM. *P ≤ 0.05, **P < 0.01 (unpaired, two-tailed t-test)