Figure 4.

Role of MCPH1 in E2F1- and adriamycin-induced apoptosis. (A,B) MCPH1 was knocked down and then rescued in doxycycline-inducible U2OS-TREX cells as in Fig 2B. Cells were then infected with AdCMV, AdE2F1, AdE2F2, AdE2F3 or AdE2F4 at an m.o.i. of 200. The cells were then cultured in a serum-free medium with 2 μg/ml doxycycline for another 48 h before collecting for annexin V staining. The data shown are the mean±s.e. of three independent experiments. ^**P=0.005 (t-test, two-tailed) compared with the siScr group; ^*P=0.02 (t-test, two-tailed) compared with the siMCPH1 group. Upper panels: Western blot analysis of the cells as described. (C) HEK293 cells were transfected with MCPH1 or a control vector. The next day, cells were treated with 5 μM adriamycin for 48 h before collection for propidium iodide staining. Apoptotic cells (sub-G1 fractions) were analysed by flow cytometry. The data shown are the mean of triplicate experiments and the P-values (two-tailed t-test). Upper panels: Western blot analysis from the cell lysates as described. (D) HEK293 cells were transfected with siScr or two different MCPH1 siRNAs (siM#1 and siM#2). The next day, cells were treated with adriamycin (5 μM) for 24 h and then measured for caspase-3/7 activity. The cell lysates were collected for immunoblottings (upper panels). HEK, human embryonic kidney cells; MCPH1, microcephalin; m.o.i., multiplicity of infection; PARP, poly(ADP-ribose) polymerase; siScr, scrambled siRNA (Liu et al, 2006); siM, MCPH1 siRNA.