Fig. 2. Global-genome repair of 5-azadC-induced DNMT1-DPCs monitored by PxP.

a Schematic depiction of the experimental workflow used to monitor the repair of 5-azadC-induced DNMT1-DPCs. Cells were synchronized via a double thymidine block and released into early/mid S-phase for 3 h prior to induction of DNMT1-DPCs by a 30-min pulse of 5-azadC. Samples were collected either immediately after 5-azadC exposure or following a chase in drug-free media. Proteasome inhibitor (MG132, 5 µM), p97 inhibitor (p97i CB-5083, 5 µM) and SUMOylation inhibitor (SUMO-E1i ML-792, 5 µM) were added 1 h prior to induction of DPCs and kept during the chase with 5-azadC-free medium. Ubiquitylation inhibitor (Ub-E1i TAK-243, 1 µM) was added together with 5-azadC. b 5-azadC-induced DNMT1-DPC formation assessed by PxP. HeLa T-REx Flp-In cells were treated as depicted in (a) with the indicated doses of 5-azadC for 30 min prior to immediate isolation of DPCs using PxP and western blotting analysis. c–f 5-azadC-induced DNMT1-DPC formation and repair upon proteasome inhibition (c), inhibition of SUMOylation (d), inhibition of ubiquitylation (e), or knock-out of RNF4 (f) assessed by PxP. HeLa T-REx Flp-In cells were treated as depicted in (a) prior to extraction of DPCs using PxP, and analysis of samples by western blotting using the indicated antibodies. g HeLa WT and RNF4 knock-out (KO) cells were treated and analysed as depicted including an optional treatment with SUMOylation inhibitor (SUMO-E1i ML-792, 5 µM), prior to extraction of DPCs using PxP and analysis of samples by western blotting using the indicated antibodies. Experiments in (c–g) were repeated three times and similar results were obtained. Source data are provided as a Source Data file.