Figure 1: S-phase defects after the inactivation of poly(ADP-ribosyl)ation.

(a) Colony formation of 3T3 MEFs of wild type (WT), PARG₁₁₀^−/− and PARP1^−/− genotype with or without HU treatment at the concentration indicated. The data represent the mean of two experiments using two independent cell lines from each genotype in duplicate. Error bars represent the s.e.m. The t-test shows significant difference of PARP1^−/− cells as compared with WT cells (#P=0.0068). (b) RDS assay of immortalized MEFs of indicated genotype and treatment. Wild type (A19), PARP1^−/− (A11), PARP1^−/− reconstituted with human PARP1 cDNA (PARP1^−/− +PARP1; A11/WT2), WT pretreated with the PARP inhibitor (PARPi, PJ-34, 10 μM) and WT cells pretreated with the Chk1 inhibitor UCN-01 (100 nM) at 2 h before UV treatment. The ratio of ³H- (after) and ¹⁴C- (before) labelled DNA was calculated after UV treatment (3 J m⁻²) and the relative DNA synthesis is shown. The data presented are the mean of two experiments using two independent cell lines from each genotype in triplicates. Error bars represent the s.e.m. (c) IB analysis of thymidine block-synchronized primary MEFs of indicated genotype wild type (WT), PARG₁₁₀^−/−, PARP1^−/− and WT pre-incubated with the PARP inhibitor (PARPi, PJ-34, 10 μM) after treatment with 2 mM HU for 3 h. Untreated cells (−), or from 0 and 3 h after HU washout were analysed for the indicated proteins or PAR. Actin was used as a loading control. (d) Co-localization of PAR and p-Chk1 foci after both HU and UV damage. Representative images (left panel) and the quantification (right panel) of co-localization of PAR and phospho-Chk1 (p-Chk1) foci in EdU-positive MEF cells after HU (4 mM) or UV (10 J m⁻²) treatment. Insets show the enlarged image of co-localization of the PAR-p-Chk1 foci. Scale bar, 5 μm. For statistical evaluation, ANOVA followed by the Newman–Keuls post hoc analysis was performed. *P<0.05; **P<0.01; ***P<0.001. NS, not significant.