Fig. 5. The ADP-ribose chromatin scars in ARH3-defective cells result from S phase DNA damage.

a Wild-type and ARH3^−/− U2OS cells were treated with DMSO vehicle or 10 μM PARPi for 24 h, washed, and then incubated in PARPi-free medium with or without 2.5 mM thymidine (THM) for the indicated time periods. ADP-ribose levels in chromatin were then measured by indirect immunofluorescence in detergent pre-extracted cells with anti-PAN-ADP-ribose binding reagent. b Cells treated as above were pulse-labeled at day 7 after release from PARP inhibitor with 10 μM EdU for the last 20 min prior to harvesting, to confirm that thymidine-induced cell-cycle arrest was successful. c Cells treated as in a were incubated on day 7 with 150 μM H₂O₂ for 10 min and ADP-ribose levels then measured, to confirm that PARP can be activated in thymidine-arrested cells. γH2AX in a, b, and c was used as a marker of replication stress in thymidine treated cells. Representative images for a, b, and c are shown. Scale bar, 20 μM. d Wild-type and ARH3^−/− U2OS cells were treated as in a and ADP-ribosylated histones detected by western blotting, using anti-PAN-ADP-ribose binding reagent.