Fig. 4

LAP2α maintains DNA replication integrity through loading RPA onto ssDNA. A DNA fiber assay with Lap2α^+/+ or Lap2α^−/− MEFs. Cells were sequentially labelled with DNA analog IdU and CldU for the indicated time followed by HU (4 mM, 4 h) treatment in the absence or presence of mirin (100 μM, 4 h). The ratios of CldU and IdU length were calculated in each treatment (n > 150). Scale bar, 10 μm. B pLenti-vector, LAP2α/wt or LAP2α/2RE stably integrated U2OS cells were transfected with control siRNA or LAP2α 3′UTR siRNA and experiments analogous to A were performed (n > 150). Scale bar, 10 μm. C Lap2α^+/+ or Lap2α^−/− MEFs were treated with 2 mM HU for 16 h and released followed by γH2AX staining and confocal microscopy inspection. The foci number of γH2AX per cell in each treatment was quantified (n > 100). Scale bar, 10 μm. D Accumulation of damaged DNA was examined and quantified with alkaline comet assay in HU (2 mM, 4 h) or CPT (1 μM, 2 h) treated Lap2α^+/+ or Lap2α^−/− MEFs (n > 150). Scale bar, 100 μm. E pLenti-vector, LAP2α/wt or LAP2α/2RE stably integrated U2OS cells were transfected with control siRNA or LAP2α 3′UTR siRNA and experiments analogous to C were performed (n > 150). Scale bar, 10 μm. F pLenti-vector, LAP2α/wt or LAP2α/2RE stably integrated U2OS cells were transfected with control siRNA or LAP2α 3′UTR siRNA followed by HU treatment and experiments analogous to D were performed (n > 300). Scale bar, 100 μm. Data are mean ± SDs for A and B from biological duplicate experiments, and C–F from biological triplicate experiments. *P < 0.05; **P < 0.01; NS, not significant; Mann-Whitney test