In vitro activity: In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168.

Cell Assay: 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°C for 10 min. After cooling on ice for 1 min, the cells are permeabilized with 90% methanol and stored at -20°C 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.