Fig. 4.

Effects of the combined hypoxia and X-ray treatments on H₂O₂ production and the antioxidant responses. A and B Representative confocal images of nuH₂O₂ and cyH₂O₂ detection in normoxic non-irradiated NSCLC cells by staining with NucPE1 and PY1-ME, respectively. Nuclei (blue) were counterstained with Hoechst 33,342. C and D Relative estimation of nuH₂O₂ and cyH₂O₂ content at 30 min and 24 h post-IR in normoxia and hypoxia. All samples were normalized and statistically compared to their respective normoxic or hypoxic control (0 Gy). Error bars are represented as SEM (n = 3) (one-way ANOVA). E Correlation between normalized (to 0 Gy) DSBs per cell and nuH₂O₂ levels for each dose in normoxia and hypoxia measured at 30 min post-IR. F Relative mRNA expression levels of intracellular NFE2L2 and CAT genes evaluated using RT-qPCR at 24 h following IR treatment in normoxia and hypoxia. The data are presented as mean of the ΔΔCt values ± SEM (n = 2, each in triplicate) (one-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001 as compared to 0 Gy normoxia; ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 as compared to 0 Gy hypoxia