Figure 4
Cdc7 is required for Aurora B activation during M-phase. (a,b) The cells from HCT116 or HCT116-323 (Cdc7 promoter mutant with reduced Cdc7 protein level) were treated with RO-3306 overnight, released and collected at indicated time. The HH3 pS28 levels were analyze by FACS (a) and the averages of three independent experiments (measured by Prism software) are shown (b). Western blotting is shown in Supplementary Fig. S4a. (c,d) The cells from AID-tagged mClover Cdc7 clone #3 or parent HCT116 were arrested with RO-3306 overnight, washed three times, incubated in medium with/without Auxin and harvested at indicated time. The HH3 pS28 levels were analyzed by FACS (c) and the averages of three independent experiments (measured by Prism software) are shown (d). Cdc7 protein levels were analyzed by Western (Supplementary Fig. S4d). (e,f) HeLa cells expressing GFP-CENP-A were cultured in glass chamber slide, arrested with RO-3306, washed and released in the presence of various drugs. Control, no drug; Cdc7 inhibitor, 10 µM PHA-767491; Aurora B inhibitor, 100 nM AZD1152. Cells were fixed and immunostained by anti-CENP-A pS7 antibody (red) and Hoechst33342 (blue) and signals were detected by LSM780 confocal microscopy (e). Imaris spot analyses were conducted (f). 0 min, n = 732; 30 min-cont, n = 665; 30 min-Cdc7i, n = 1049; 30 min-Bi, n = 322; 60 min-cont, n = 680; 60 min-Cdc7i, n = 837; 60 min-Bi, n = 648; **p < 0.0001.
