Fig. 2.

Combination therapy with olaparib and AZD1775 induced more potent DNA damage and apoptosis. (A) Combination effect was investigated by MTT for 5 days before treatment with olaparib (0, 0.01, 0.1, 1, and 10 μM) and AZD1775 (0, 0.001, 0.01, 0.1, and 1 μM), dose ratio was 10:1. CI value was analyzed by CalcuSyn software. If the value is lower than 1 is synergistic, and the cell lines that higher than 1 exhibit an antagonistic effect. (B) Colony-forming analysis was performed with olaparib (0, 1, 5 μM), AZD1775 (0, 0.01, 0.05, 0.1 μM) or both for 10 days. Error bars represent mean±standard deviation (SD) (n=3). (C) Comet analysis was conducted after treatment with olaparib (5 μM), AZD1775 (0.2 μM) or both for 72 hours. The tail intensity and tail moment were analyzed using the Comet assay IV program. The experiment was repeated three times. Data are expressed as mean±standrad error of mean (n=100). (D) Apoptosis analysis was performed using annexin V/propidium iodide (PI) double staining after 24-hour and 72-hour treatment with olaparib (5 μM), AZD1775 (0.1 μM) or both. Error bars represent mean±SD (n=4). (E) Cell cycle analysis were performed with PI-stained BTC cells using flow cytometry after 72-hour treatment with olaparib (5 μM), AZD1775 (0.2 μM) or both. Error bars represent mean±SD (n=3). (F) Western blotting of two cell lines was performed after 72-hour incubation with olaparib, AZD1775, or both. *p < 0.05, **p < 0.01, ***p < 0.001.