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. 2020 Mar 5;16(9):1536–1550. doi: 10.7150/ijbs.40042

Figure 5.

Figure 5

Combination of icotinib and BDMC induces marked DNA damage that depend on autophagy and oxidative stress. (A) H460 cells were treated with icotinib and/or BDMC, or the two combinations plus 3-MA at indicated doses for 48h, then subjected to immunoblot assay to determine phosphorylated (p) or total protein expressions as indicated. (B) H460 cells were treated with icotinib plus BDMC at indicated doses for 12, 24, and 48 h, or icotinib plus BDMC in combination with NAC at indicated doses for 48 h, and then subjected to immunoblot assay to detect the proteins expressions as indicated. (C and D) H460 and H1781 cells were treated with icotinib and/or BDMC, or the two drug combination plus 3-MA at indicated doses for 48 h. Then comet assay was performed to detect DNA double strand breaks, and images show detectable comet tail when visualized under a fluorescent microscopre. Tail moment in the cells were quantified using Comet Score software version 1.5. *P < 0.05 vs icotinib + BDMC + 3-MA, or BDMC. (E and F) H460 cells were treated with icotinib or BDMC alone, or icotinib plus BDMC, or the two combination plus NAC at indicated doses for 12 h, then colocalizations of p-ATM and 53-BP1 foci were determined by immunofluorescent staining using specific antibodies. Frequency of positive colocalization (the percentages of cells exhibiting more than 10 foci in the nucleus on confocal images) was analyzed by SlideBook 5.5 software. (*P < 0.01 versus icotinib + BDMC + NAC, or BDMC).