Figure 7.

Elevated Stat3 expression is involved in the sensitivity of NiT cells to autophagy. The basal expression levels of phosphorylated and total Stat3 in BEAS-2B and NiT cells were evaluated by Western blot analysis (A). NiT cells were incubated with Ni²⁺ (2 mm) for 24 h in the presence or absence of stattic (10 μm), and the LC3 levels and the number of cells containing GFP-LC3 puncta were assessed by Western blot analysis and fluorescence microscopy, respectively (B). NiT cells were transfected with siRNA to knock down Stat3; the levels of LC3 were assessed by Western blot analysis, and the number of cells containing GFP-LC3 puncta were determined by fluorescence microscopy (C). BEAS-2B cells were transfected with a Stat3 plasmid, and the overexpression of Stat3 was confirmed by Western blot analysis (D). The Stat3-overexpressing BEAS-2B cells were exposed to various concentrations of Ni²⁺ (0–2 mm) for 24 h, and the levels of LC3 (E) or the number of cells containing GFP-LC3 puncta (F) were evaluated by Western blot analysis and fluorescence microscopy, respectively. To investigate the relationship between Stat3 and Nrf2, NiT cells were transfected with Nrf2-specific siRNA. After a 12-h transfection, the expression levels of phosphorylated Stat3 and total Stat3 were assessed by Western blot analysis (G). In addition, chromatin was immunoprecipitated from BEAS-2B and NiT cells with an anti-Nrf2 antibody. Nucleotide sequence analysis of the 3-kb Stat3 promoter revealed the presence of five consensus AREs (H). The binding of Nrf2 to the Stat3 promoter was assessed by normal real-time PCR (I) or quantitative real-time PCR (J) with primers specific for the ARE-containing regions of the promoter. Graphic data are the mean ± S.E. of three independent experiments, with significant differences indicated as *, p < 0.05; **, p < 0.01, and ***, p < 0.001, determined by ANOVA and Scheffe's test. GAPDH and β-actin were used as loading controls.