Cell experiment:

Cells are treated with different concentrations of the PRT4165 60 min prior to irradiation (2 Gy). The cells are harvested 2 h after IR. The cells are washed with PBS twice and then fixed with 1% paraformaldehyde at 37℃ for 10 min. After cooling on ice for 1 min, the cells are permeabilized with 90% methanol and stored at -20℃ overnight. Fixed cells are washed with PBS twice and blocked with FACS incubation buffer (0.5% BSA in PBS) for 10 min. The cells are then stained with anti-phosphohistone H3 (serine 10) antibody at 1:500 dilution in FACS incubation buffer for 1 h at room temperature[1].

PRT4165 is a potent inhibitor of PRC1-mediated H2A ubiquitylation.

PRT4165 is a potent inhibitor of PRC1-mediated H2A ubiquitylation. In vitro E3 ubiquitin ligase activity assays reveal that PRT4165 inhibits both RNF2 and RING 1A, but not RNF8 nor RNF168. In the presence of PRT4165, H2A ubiquitylation could be completely inhibited regardless of whether RING1 or RNF2 contributes the E3 ubiquitin ligase activity. Treatment of cells for 60 min with 50 μM PRT4165 results in a dramatic reduction in total ubiquitylated histone H2A. It is also found that longer exposure of the cells with the PRT4165 (30 and 60 min) leads to increased levels of γ-H2AX in unirradiated cells. PRT4165 inhibits double-strand break (DSB) repair at the 8-h time point compare with mock treated cells. Cells treated with increasing concentrations of PRT4165 show increasing numbers of cells in G2/M[1].

References:
[1]. Ismail IH, et al. A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks. J Biol Chem. 2013 Sep 13;288(37):26944-54.