Fig. 6: HMGB3 prevents PARP1 trapping and confers DNA repair upon olaparib treatment.

A A2780 and UWB1.289 cells transfected with PLKO.1 and HMGB3 shRNA (shHMGB3) were treated with olaparib (A2780, 4 µM; UWB1.289, 2 µM) for 4 h prior to incubation with 4 mM MMS for 20 min. Western blot was conducted to detect the protein levels of PARP1 in the chromatin-bound fractions. B Quantification of the PARP1 protein levels in (A). C, D A2780 and UWB1.289 cells transfected with PLKO.1, HMGB3 shRNA (shHMGB3), PCMV, and PCMV HMGB3 were treated with olaparib (A2780, 4 µM; UWB1.289, 2 µM) for 72 h. Rad51 and γH2AX foci were examined by immunofluorescence staining. Scale bar: 10 µm. Quantification of the number of γH2AX and RAD51 foci (>10) can be found in Fig. S6B. E A2780 and UWB1.289 cells transfected with PLKO.1 and HMGB3 shRNA (shHMGB3) were treated with olaparib (A2780, 10 µM; UWB1.289, 5 µM) for 24 h. After that, olaparib was removed and fresh drug free media was added. Cells were cultured further and γH2AX foci were observed at 12 h, 24 h, 36 h, and 48 h post drug removal. F, G A2780 and UWB1.289 cells transfected with PLKO.1, HMGB3 shRNA (shHMGB3), PCMV, and PCMV HMGB3 were treated with olaparib (A2780, 2 µM and 4 µM; UWB1.289, 1 µM and 2 µM) for 72 h. The protein level of γH2AX was detected by western blot. H, I Quantification of the protein level of γH2AX in (F) and (G), respectively. (Data are presented as the mean ± SEM, *p < 0.05, **p < 0.01, n = 3).