FIGURE 6.

The NRF2/HMOX-1 pathway plays a role in CTX-induced ferroptosis. (A) Relative Nrf2, Hmox-1 and Slc7a11 mRNA levels in 4T1 cell lines treated with 4HC (20 μM) for the indicated times. Data are represented as the means ± SD from three independent experiments. ns, not significant, ^** p < 0.01, and ^*** p < 0.001 (one-way ANOVA analysis). (B) Western blot analysis of NRF2/HMOX-1 system expression in 4HC-treated cells. Histone H3 served as a loading control, and representative results from three experiments are shown. (C) Images of immunofluorescence staining for NRF2. Representative images from experiments with similar results are shown (scale bar = 100 μm). (D) Intracellular GSH levels and the viability of cells after exposure to the specific HMOX-1 inhibitor zinc protoporphyrin (ZNPP) and agonist Hemin. Values are presented as the means ± SD from more than three independent experiments. ***p < 0.001 (one-way ANOVA analysis). (E) Western blot analysis of NRF2, HMOX-1 and SLC7A11 levels in transfected cells. GAPDH served as a loading control, and representative results from three experiments are shown. (F) Flow cytometry analysis of the MMP in cells treated under the indicated conditions; representative results from three experiments are shown.