Figure 4
miR-134 enhances DNA repair efficiency by promoting H2AX S139 phosphorylation. (a) Left, representative comet assay revealing the formation of DNA breaks (formation of a ‘comet tail’) in miR-134-transfected T29 and SKOV3 cells treated with adriamycin (0.5 μM) for 24 h and then allowed to recover for 12 h. Right, box plot graph showing Tail DNA%. (b) ChIP assay with anti-γ-H2AX was performed 24 h after SKOV3-Hygro-EGFP cells were co-transfected with miR-134 or the control mimics. The genomic locations of the qPCR primers are referred to as ChIP primers in Supplementary Figure S6a. (c) The proportion of EGFP-positive cells was determined 48 h after SKOV3-Hygro-EGFP cells were co-transfected with the pCBASceI plasmid and miR-134 or the control mimics. (d) Representative western blot images of γ-H2AX and H2AX in H2AX-S139A-expressing cells or control cells 24 h after adriamycin (0.5 μM) treatment. (e) Representative western blot images of γ-H2AX and H2AX in cells co-transfected with the H2AX-S139A plasmid and miR-134 or the control mimics for 48 h. (f) The box plot graph showing tail DNA% in H2AX-S139A-transfected cells treated with adriamycin (0.5 μM) for 24 h and then allowed to recover for 12 h. (g) ChIP assay using anti-γ-H2AX was performed 24 h after SKOV3-Hygro-EGFP cells were co-transfected with pCBASceI and the H2AX-S139A plasmid or the control plasmid. (h) The proportion of EGFP-positive cells was determined 48 h after SKOV3-Hygro-EGFP cells were co-transfected with the pCBASceI plasmid and the H2AX-S139A plasmid or the control plasmid. (i) The box plot graph depicting tail DNA% in T29 cells co-transfected with miR-con/miR-134 and pcDNA3.1/H2AX-S139A after a 24 h adriamycin treatment and a 12-h recovery. (j) The proportion of EGFP-positive cells was determined 48 h after SKOV3-Hygro-EGFP cells were co-transfected with miR-con/miR-134 and pcDNA3.1/H2AX-S139A. Data are shown as mean±S.D. from three independent experiments. *P<0.05, **P<0.01 by Student’s t-test
