Fig. 3: The effects of 3-methyladenine (3-MA) on TGF-β1-induced autophagy activation and epithelial-to-mesenchymal transition (EMT) in HPMCs.

A TGF-β1 (2 and 5 ng/mL) induced autophagy activation but co-treatment with 3-MA (2 mM; 3-MA + T2 and 3-MA + T5) decreased autophagy activation. This was confirmed using a Cyto-ID Autophagy Detection Kit. B, C 3-MA treatment (2 mM) suppressed TGF-β1 (2 and 5 ng/mL) and induced autophagy activation. This was confirmed by western blotting analysis, which revealed decreases in Beclin 1, LC3B, and ATG5, and an increase in p62 levels. Protein levels of the mesenchymal markers fibronectin and α-SMA were decreased by 3-MA in TGF-β1-treated HPMCs. D Representative immunofluorescence images showing LC3 staining of TGF-β1-induced autophagy activation and EMT in HPMCs. (D, upper phase) Representative immunofluorescence images showing LC3B staining of TGF-β1-induced autophagy activation. Positive control cells were treated with 30 µM chloroquine for 16 h. Arrows indicate autophagic flux. (D, lower phase) Representative immunofluorescence images showing α-SMA staining of TGF-β1-induced EMT. Arrows indicate the lamellipodia. Scale bar = 40 μm. The 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 < 0.001 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.