Fig. 4

Inhibitory effects of GPX2 and CYGB expression on DNA lesions caused by radiation in HMECs. (a) GPX2 and CYGB mRNA expression levels measured by RT–qPCR. Data are the means and SEs of at least three independent assays. (b) Detection of GPX2 and CYGB protein expression in HMECs by Western blotting analysis. (c) The localization of GPX2 and CYGB proteins inside cells. CD49f was used as a HMEC marker. Scale bar, 10 μm. (d) Determination of double-strand breaks (DSBs) using a neutral comet assay. The left panel shows the %tail DNA calculated from the comet tails shown in the right panel (n = 100). (e) Measurement of γH2AX foci observed in the nucleus at 4 h post-irradiation (n = 50). The left panel shows the numbers of γH2AX foci per cell. The right panel shows IF images of γH2AX foci, where green, red, and blue indicate γH2AX, ΔNp63, and DAPI, respectively. In the bottom images, DAPI was used as a counterstaining dye instead of ΔNp63. (f) FCM detection of intercellular reactive oxygen species (ROS) generated in HMECs immediately after X-irradiation. DCFH-DA, one of the major DCF derivatives, and PI were used as probe dyes for detecting ROS and dead cells, respectively. Data are the means and SEs of at least three independent assays. *P < 0.05, **P < 0.01 by Student’s t test