Figure 4.

Nrf2 plays a critical role in reducing apoptosis and increasing autophagy in NiT cells. The basal expression levels of Nrf2 in BEAS-2B and NiT cells were assessed by Western blot analysis (A). BEAS-2B and NiT cells were exposed to various concentrations of Ni²⁺ (0–2 mm) for 24 h (B), or to Ni²⁺ (2 mm) for various lengths of time (0–24 h) (C), and the levels of Nrf2, NQO1, HO-1 were assessed by Western blot analysis. To decrease Nrf2 levels, BEAS-2B and NiT cells were transfected with Nrf2-specific siRNA. After overnight transfection, cells were exposed to Ni²⁺ (2 mm) for an additional 24 h. Nrf2 expression levels were evaluated by Western blot analysis (D), and an apoptosis assay was performed after staining with annexinV/PI (E). In addition, the levels of LC3, PARP, and caspases 3 and 7 were assessed by Western blot analysis (F), and the number of cells containing GFP-LC3 puncta was quantified (G). Data presented graphically are the mean ± S.E. of three independent experiments, with significant differences from controls indicated as *, p < 0.05; **, p < 0.01, and ***, p < 0.001, identified by ANOVA and Scheffe's test). GAPDH was used as loading control in the Western blot analyses.