Knock-down of p53 in AE cells increases resistance to DNA damage and interferes with the G1 checkpoint. (A) RQ-PCR analysis shows decreased TP53 gene expression in AE cells expressing shRNA targeting p53 (AE-p53sh) compared with AE cells expressing scrambled sequence (AE-scrambled). (B) Western blot analysis shows reduction of total p53 and p21 proteins and decreased level of induction after irradiation in AE-p53sh compared with AE-scrambled cells. Cells were irradiated (5 Gy) and samples were analyzed after 5 hours. (C) The AE-p53sh cells have reduced induction of p53 target genes after irradiation. Bars represent a ratio of the target gene expression in irradiated to nonirradiated samples. (D) BrdU incorporation demonstrates a faulty G1 checkpoint in AE-p53sh compared with AE-scrambled and as well as in Kasumi-1 cells after irradiation. (E) Staining for early mitotic antigens using MPM-2 antibodies demonstrates intact initiation of the G2 checkpoint in all cultures. (F,G) The p53 knockdown increases resistance of AE cells to ionizing radiation and to AraC. Proliferation assays were performed at 72 hours after irradiation or after 72 hours of incubation with AraC. Experiments were repeated at least twice with similar results. Error bars represent SD, and numbers on plots are percentages of total cells.