Fig. 5

LAP2α-promoted RPA loading is required for ATR activation and homologous recombination. A Immunoblotting analysis of ATR kinase activity. Control U2OS cells and LAP2α/wt or LAP2α/2RE stably integrated U2OS cells were transfected with indicated siRNAs and in the absence or presence of CPT (1 μM, 1 h). The cellular extracts were collected to examine CPT-induced phosphorylation events. B DNA fiber assay with Lap2α^+/+ or Lap2α^−/− MEFs. Cells were sequentially labelled with DNA analog IdU and CldU for the indicated time. Fork speed (n > 120) and fork symmetry (n > 25) were determined by measuring the length of CldU track, and the percentage of new origins was quantified with only CldU staining fibers. C Immunostaining and confocal microscopy analysis of RAD51 foci formation. LAP2α/wt or LAP2α/2RE stably integrated U2OS cells were transfected with LAP2α 3′UTR siRNA, exposed to IR (4 Gy) and cultured for 3 h followed by 1 h EdU labelling before collection. The foci number of RAD51 per EdU-positive cell in each group was quantified (n > 150). D Homologous recombination efficiencies monitored by DR-GFP reporter assays. Control DR-GFP U2OS cells and DR-GFP U2OS cells that allow for Dox-inducible expression of stably integrated pTRE-LAP2α/wt or pTRE-LAP2α/2RE were co-transfected with HA-I-SceI and the indicated siRNAs. Cells were treated with vehicle or Dox (1 ng/ μl) for 48 h to induce the expression of LAP2α/wt or LAP2α/2RE. The proportions of GFP-positive cells were determined by flow cytometry. E Survival analysis of U2OS cells expressing LAP2α 3′UTR siRNA and LAP2α/wt or LAP2α/2RE under different drug treatment. Data are mean ± SDs for B from biological duplicate experiments, and C–E from biological triplicate experiments. **P < 0.01; NS, not significant; Mann-Whitney test for B and C; one-way ANOVA for D; two-way ANOVA for E. Scale bar, 10 μm