Fig. 6

Effect of superoxide anion on EMT. a H460 cells were incubated with 5 µM DMNQ (superoxide anion inducer) or 50 µM MnTBAP (superoxide anion inhibitor), in the presence or absence of zinc (50 µM) for 24 h prior to morphology examination using phase contrast microscope; scale bar 100 µm. b Cells were treated with 5 µM DMNQ or 50 µM MnTBAP in the presence or absence of zinc (50 µM) for 24 h. Expression of vimentin were analyzed by immunofluorescence staining; scale bar 100 µm. c Cells were treated with 5 µM DMNQ or 50 µM MnTBAP, in the presence or absence of zinc (50 µM) for 24 h. The cells were collected and analyzed for EMT markers by western blotting. The blots were re-probed with β-actin to confirm equal loading. d The immunoblot signals were quantified by densitometry and mean data from independent experiments were normalized to the results. The bars are the mean ± SD of independent triplicate experiments. *p < 0.05 versus untreated control cells. ^#p < 0.05 versus 50 µM zinc-treated cells. e Cells were incubated with DHE at 4 °C for 30 min prior to the treatment of DMNQ (5 µM) or zinc (50 µM), and the fluorescence intensity was analyzed by flow cytometry at 3 h. Mean intensity was normalized to untreated control cells and represented as relative superoxide anion levels. Values are means of independent triplicate experiments ± SD. *p < 0.05 versus untreated control