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Fig. 3 | Genome Biology

Fig. 3

From: LAP2α preserves genome integrity through assisting RPA deposition on damaged chromatin

Fig. 3

LAP2α promotes the loading of RPA onto damaged chromatin. A Proteins associated with replication forks were isolated by iPOND as described in “Methods” and detected by immunoblotting. Lap2α+/+ or Lap2α/ MEFs were EdU-labelled for 10 min and harvested immediately or after a 1-h chase with 2 mM HU. B Quantitative analysis of Rpa1, Rpa2, and Pcna binding to EdU-labelled nascent replication forks with iPOND assays. The relative intensity of chromatin bound factors to histone H3 was normalized against that in vehicle-treated control cells. C High content imaging analysis of foci intensity of RPA1 and RPA2 in U2OS cells. pLenti-vector, LAP2α/wt or LAP2α/2RE stably integrated U2OS cells were transfected with control or LAP2α 3′UTR siRNA (siLAP2α-2) and treated with 2 mM HU for 4 h or 1 μM CPT for 2 h followed by pre-extraction, fixation, immunostaining, and high content microscopy analysis. Before collection, cells were labelled with EdU for 1 h. D Representative images from C collected by confocal microscopy. E Laser micro-irradiation (IR) (50% laser energy) followed by live cell imaging analysis of GFP-RPA1 recruitment kinetics in GFP-RPA1 expressing Lap2α+/+ or Lap2α/ MEFs. Fluorescence intensities in micro-irradiated areas relative to the nuclear background were quantified (n > 20). F pLenti-vector, LAP2α/wt or LAP2α/2RE stably integrated U2OS cells were co-transfected with DsRed-RPA1 and control siRNA or LAP2α 3′UTR siRNA followed by Hoechst sensitization, micro-IR (405 nm) and live cell imaging analysis. Fluorescence intensities in micro-irradiated areas relative to the nuclear background were quantified (n > 20). Data are mean ± SDs for B, E, and F from biological triplicate experiments, and C from biological duplicate experiments. **P < 0.01; NS, not significant; one-way ANOVA for B; Mann-Whitney test for C; two-way ANOVA for E and F. Scale bar, 10 μm

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