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Fig. 5 | Journal of Biomedical Science

Fig. 5

From: O6-methylguanine-DNA methyltransferase modulates cisplatin-induced DNA double-strand breaks by targeting the homologous recombination pathway in nasopharyngeal carcinoma

Fig. 5

MGMT inhibition suppressed HR activity in NPC cells. The representative images of immunofluorescence foci of γ-H2AX and RAD51 in a HONE-1 and b TW01 cells are shown. Following treatment with O6BG (60 μM), CDDP (10 µM), or a combination of both for 24 h, the immunofluorescence foci of γ-H2AX and RAD51 were captured at ×60 on a confocal immunomicroscope. DAPI was used to detect the nuclei. Representative images of at least three independent experiments are shown. c The percentages of γ-H2AX-positive NPC cells in a and b, indicating the levels of DSB formation, were quantified in response to drug treatment (left panel). Only cells with more than five foci of γ-H2AX were considered positive. The percentages of γ-H2AX-positive cells with the recruitment of RAD51 foci in a, b were quantified in terms of DNA repair activity (right panel). Following CDDP or combination treatment, cells with more than five colocalized foci of γ-H2AX and RAD51 were considered positive. At least 100 cells per sample were counted in each experiment. d The representative images of immunofluorescence foci of γ-H2AX and RAD51 in MGMT-proficient or -deficient HONE-1 cells are shown. After cells were transfected with scrambled or MGMT-targeted siRNA for 24 h, these cells were treated with CDDP for another 24 h. The immunofluorescence foci of γ-H2AX and RAD51 were detected using confocal immunomicroscope as indicated in a. Scale bars indicate 10 µm. e The levels of DNA damage (upper panel) and DNA repair activity (low panel) in d were quantified by measuring the immunofluorescence foci of γ-H2AX and RAD51as indicated in c. Following treatment with the f indicated concentration of O6BG or g transfection of MGMT-targeted siRNA for 24 h, HR activities in the tested cells were determined using a plasmid-based recombination reporter assay. The value of the control cells was set as 100% after internalization with a backbone plasmid. Bar values are represented as mean ± SD of at least three independent experiments. *P < 0.05

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