Fig. 6

LAP2α is engaged into damaged chromatin in a PARP1-dependent manner. A Laser micro-IR (35% laser energy) followed by live cell imaging analysis of DsRed-LAP2α recruitment kinetics in PARP1 inhibitor (PARPi)-treated U2OS cells. DsRed-LAP2α expressing cells were pre-treated with different PARPis (10 μM) for 4 h before laser micro-IR. Fluorescence intensities in micro-irradiated areas relative to the nuclear background were quantified (n > 20). B Laser micro-IR (50% laser energy) followed by live cell imaging analysis of DsRed-LAP2α recruitment kinetics in PARP1-knockdwon or -overexpression U2OS cells. pLenti-vector, PARP1/wt, or PARP1/E988K stably integrated U2OS cells were co-transfected with control siRNA or PARP1 3′UTR siRNA (siPARP1-1) and DsRed-LAP2α. Fluorescence intensities in micro-irradiated areas relative to the nuclear background were quantified (n > 20). C U2OS-LacO cells expressing mCherry-LacI, PARP1 variants, and PARP1 3′UTR siRNA were labelled with EdU for 1 h before immunostaining and confocal microscopy analysis. The intensity of LAP2α foci in mCherry-LacI and EdU-positive cells was quantified and normalized to the nuclear background (n > 100). D ssDNA-pull-down analysis of LAP2α accumulation, PARP1 binding, and RPA loading with nuclear extracts from LAP2α-knockdown U2OS cells under rucaparib (10 μM, 4 h) treatment. 5’ biotin-labelled 70-nt ssDNA was used in pull-down assays. E Co-immunoprecipitation analysis of the interaction between LAP2α and PARP1 with cellular extracts from U2OS cells pre-treated with vehicle or rucaparib (10 μM, 4 h). F A proposed regulatory model for PARP1 in directing LAP2α-promoted RPA loading at damaged chromatin. X-factor represents the molecular machinery that may function to retain the longer occupancy of LAP2α on damaged chromatin. Data are mean ± SDs for A–C from biological triplicate experiments. **P < 0.01; NS, not significant; two-way ANOVA for A and B; Mann-Whitney test for C. Scale bar, 10 μm