Figure 7.

PARP1 inhibition delayed DNA repair and leads to prolonged cell cycle arrest at G2/M. (A) Both the cell lines SiHa and ME180 were treated with 10 µM cisplatin and/or olaparib for 24 hrs. After 24 hrs drug was removed (DR0) and fresh drug free media was added. Cells were cultured further and γH2A.X foci were observed very 24 hr till 10 days post drug removal (DR0 to DR10). Cells with >20 γH2A.X foci to 0 foci were counted in blind fashion and at least 200 cells per group in several different 60× objective fields were acquired. Quantitative plot displaying distribution of γH2A.X foci in three treatment groups in both the cell lines. [Time point DR3, frequency of cells with more than 20 foci per cell in SiHa; cisplatin vs. cisplatin + olaparib *p = 0.0467, at DR4; cisplatin vs. cisplatin + olaparib *p = 0.0268, at DR5; cisplatin vs. cisplatin + olaparib **p = 0.0060 and frequency of cells with more than 20 foci per cell in ME180 at DR3; cisplatin vs. cisplatin + olaparib *p = 0.0390, at DR4; cisplatin vs. cisplatin + olaparib *p = 0.041, at DR5; cisplatin vs. cisplatin + olaparib **p = 0.0024]. (B) Both the cell lines SiHa and ME180 were treated as indicated and cell were stained with FITC tagged anti γH2A.X & co-stained with PI and analysis was done using FACS for DNA damage in each cell cycle phases. Graph showing the percentage of cells with γH2A.X foci in SiHa and ME180 cell lines. PARP1 inhibition in cisplatin treated cells shows significant accumulation of DNA damage in late S and G2/M phase compared to G1. (C) Graph showing frequency of γH2A.X foci resolution in PARP1 silenced and control counterpart after cisplatin treatment. PARP1 silenced cells displayed significant delay in γH2A.X foci resolution after drug removal. (D) Similarly, PARP inhibition along with cisplatin treatment for 48 hrs shows significant & prolonged G2/M and late S phase arrest in both the cell lines after drug removal as in previous experiments.