Figure 4

KU60019 increases the level of the TOP2β-DNA cleavage complex and impairs the repair of VP-16-induced DSBs. (A, B) H1299 cells were treated with VP-16 alone or in conjunction with KU60019 for the appropriate times, and then the TOP2cc levels were determined by FACS. (C, D) Two hours were spent treating H1299 cells with VP-16 or VP-16 in conjunction with KU60019 (5 μM). The cells were then collected for the TARDIS assay. (E, F) H1299 cells were pretreated with KU60019 (5 μM) for 1 h and then cotreated with VP-16 for an additional 1 h. Cells were then harvested for the neutral comet assay. Representative images are shown, and the data are presented as the mean ± S.D. from three independent experiments; ***p < 0.001. (G) Cells treated with VP-16 alone or in conjunction with KU60019 for the indicated time periods were harvested at the indicated time points for immunofluorescence. (H) H1299 and A549 cells were treated with KU60019 or VP-16 alone or in conjunction with IB with the indicated Abs. All experiments were independently repeated three times. According to the molecular weight, the nitrocellulose membrane was cut prior to hybridization with antibodies and the original blots are presented in Supplementary Fig. 4. Molecular weight markers are noted to the left of blot figure.