Fig. 4: The effects of NOX4 inhibition on TGF-β1-induced autophagy activation and epithelial-to-mesenchymal transition (EMT) in HPMCs.

A The expression of NOX4 and autophagy marker protein levels following GKT137831 (20 μM; GKT20) and TGF-β1cotreatment. B GKT137831 (20 μM) co-treatment resulted in lower NOX4 protein levels than TGF-β1 (5 ng/mL) treatment alone. Cotreatment with GKT137831 (20 μM) suppressed TGF-β1-induced autophagy activation. This was confirmed by western blotting analysis, which revealed decreased Beclin 1, LC3B, and ATG5, and increased p62 levels. The protein levels of the mesenchymal markers fibronectin and α-SMA were decreased by GKT137831 in TGF-β1-treated HPMCs. C TGF-β1 (2 and 5 ng/mL) induced autophagy activation and co-treatment with GKT137831 (20 μM; GKT20 + T2 and GKT20 + T5) decreased autophagy activation. This was confirmed using a Cyto-ID Autophagy Detection Kit. D Representative immunofluorescence images showing LC3B staining of TGF-β1-induced autophagy activation (upper phase). Positive control cells were treated with 30 µM chloroquine for 16 h. The arrows indicate autophagic flux. Representative immunofluorescence images showing α-SMA staining of TGF-β1-induced EMT (lower phase). The arrows indicate the lamellipodia. Scale bar = 40 μm. Data are presented as mean ± standard error (SE). n = 4 per group. ^*P < 0.05 vs. control; ^**P < 0.01 vs. control; ^***P < 0.001 vs. control; ^#P < 0.05 vs. TGF-β1 2 ng/mL; ^##P < 0.01 vs. TGF-β1 2 ng/mL; ⁺P < 0.05 vs. TGF-β1 5 ng/mL; ⁺⁺P < 0.01 vs. TGF-β1 5 ng/mL; and ⁺⁺⁺P < 0.001 vs. TGF-β1 5 ng/mL.