Fig. 3
PARP1 and PARP2 function in the resolution of replication-associated damage independently of BER. a U2OS cells were treated with 1 μM EdU and 0.25 mM MMS for 1 h, before pre-extraction and fixation. EdU and γ-H2AX were detected by immunofluorescence. Representative images of EdU-positive and EdU-negative nuclei are shown. Scale bars represent 10 μm. b The indicated cell lines were treated as in a and following immunofluorescence γ-H2AX foci quantified in G1/G2 phase (EdU-negative) and S-phase (EdU-positive) cells. Error bars represent the SEM from four independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (*** p < 0.001). c The indicated cell lines were treated with 1 μM EdU (Unt), or with 1 μM EdU and 0.25 mM MMS for 1 h before recovery in fresh media, and γ-H2AX foci quantified by immunofluorescence in EdU-positive cells. Times indicated show hours since addition of EdU and MMS. Error bars represent the SEM from four independent experiments. Statistical significance was determined as in b. d U2OS cells transfected with control or XRCC1 siRNA, or parp1/2Δ cells were treated as in c and γ-H2AX foci quantified in EdU-positive cells (lower panel). Knockdown of XRCC1 in U2OS cells was confirmed by Western blotting using the indicated antibodies (upper panel). Times indicated show hours since addition of EdU and MMS. Error bars represent the SEM from four independent experiments. Statistical significance was determined as in b. e The indicated cell lines transfected with XRCC1 siRNA were treated as in c and γ-H2AX foci quantified in EdU-positive cells. Times indicated show hours since addition of EdU and MMS. Error bars represent the SEM from three independent experiments. Statistical significance was determined as in b
