Fig. 4.
Synthetic lethal interaction between disablement of the CPPC and overexpression of Myc. (A) Cell killing by AZD1152. RPE-NEO cells (□ and ■) and RPE-MYC cells (○ and ●) were treated with 50 nM AZD1152 for 3 d and then either transferred into drug-free medium (○ and □) or maintained in fresh medium containing VX-680 (● and ■). Cells were harvested at the indicated intervals and then assayed for viability by the trypan blue exclusion assay. The data are expressed as mean ± SD for three independent experiments. The arrow indicates the time at which VX-680 treatment was terminated. (B) Depletion of the chromosomal passenger proteins survivin, aurora-B, and INCENP by RNAi. Extracts from RPE-MYC cells treated with RNAi against luciferase, INCENP, surviving, or aurora-B were analyzed by immunoblotting for expression of survivin, aurora-B, and INCENP at 4 d after transfection with RNAi. (C) Early apoptosis elicited by depletion of chromosomal passenger proteins in cells that overexpress Myc. Apoptosis was assessed by DAPI staining of DNA at 4 d after transfection of RPE-NEO cells (□) and RPE-MYC cells (■) with RNAi against the indicated genes. Cells with fragmented nuclei were scored as positive for apoptosis. Each column, here and in D, represents the average of three independent experiments, and each experiment was done in triplicate. Error bars represent 1 SD. (D) Autophagy elicited by depletion of chromosomal passenger proteins in cells that overexpress Myc. Autophagy was assessed by immunostaining for LC3 at 7 d after transfection of RPE-NEO cells (□) and RPE-MYC cells (■) with RNAi against the indicated genes. Cells that had more than 40 LC3 puncta per cell were scored as positive. (E and F) Delayed cell death elicited by depletion of the chromosomal passenger proteins. RPE-MYC cells (E) and RPE-NEO cells (F) were replated at 5% and 20% confluence, respectively, at 4 d after transfection with RNAi against indicated genes. The depletions by RNAi were comparable to those illustrated in B. Cells were photographed after being fixed and stained with crystal violet at 4 d (E) or 7 d (F) after replating.