Figure 4.

(A) Clonogenic survival assay for AZD1775 in 231control and XRCC1_KO cells untreated or pre-treated with olaparib (5 μM). (B) Quantification of γH2AX levels by flow cytometry in 231control, 231 (XRCC1_KO) and 157 cells treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM) for 24 h. (C) Quantification of cell cycle progression by flow cytometry in 231control, 231 (XRCC1_KO) and 157 cells treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM) for 24 h. (D) Quantification of apoptotic cells by annexin V flow cytometry in 231control, 231 (XRCC1_KO) and 157 treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM) for 24 h. Cells were plated overnight then treated with 5 μM of olaparib or left untreated for 24 h. The next day, untreated and olaparib pre-treated cells were treated with 10 μM of AZD1775 for another 24 h. After incubation cells were collected by trypsinization and stained for flow cytometry analysis as described in the Methods section. (E) Clonogenic survival assay for HeLa control and HeLa (XRCC1_KD) cells untreated or olaparib pre-treated (5 μM) in different doses of AZD1775. (F) Quantification of γH2AX levels by flow cytometry in HeLa control and (XRCC1_KO) cells treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM) for 24 h. (G) Quantification of cell cycle progression by flow cytometry in HeLa control and (XRCC1_KO) cells treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM) for 24 h. (H) Quantification of apoptotic cells by annexin V flow cytometry in HeLa control and (XRCC1_KO) cells treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM) for 24 h. Cells were plated overnight then treated with 5 μM of olaparib or left un-treated for 24 h. The next day, untreated and olaparib pre-treated cells were treated with 10 μM of AZD1775 for another 24 h. After incubation cells were collected by trypsinization and stained for flow cytometry analysis as described in the Methods section. (I) Representative photo micrographic images of 231 control, 231 (XRCC1_KO), 157 cells, HeLa control and HeLa_XRCC1_KD 3D-spheroids treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM). (J) Quantification of spheroid size in 231control, 231 (XRCC1_KO) and 157 treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM). (K) Quantification of viable, dead cells by flow cytometry in 231control, 231 (XRCC1_KO) and 157 treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM). (L) Representative photo micrographic images of HeLa control and (XRCC1_KO) cells treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM). (M) Quantification of spheroid size in HeLa control and HeLa (XRCC1_KO) treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM). (N) Quantification of viable, dead cells by flow cytometry in HeLa control and HeLa (XRCC1_KO) treated with AZD1775 (10 μM) or treated with AZD1775 (10 μM) plus olaparib (5 μM).

*p ⩽ 0.05; **p ⩽ 0.01; *** p ⩽ 0.001.