Fig. 2.

TGF-β3 activated autophagy of airway epithelial cells. (a) Representative immunohistochemistry of TGF-β3 proteins in lung tissues from PBS-mice, OVA-mice, 3-MA-mice and 3-MA-OVA-mice. (b) TGF-β3 in BALF was detected by ELISA. (c-d) 16HBE cells were treated with different concentrations of TGF-β3 for 24 h. Autophagy and target proteins were detected by western blotting in 16HBE cells. (e-f) 16HBE cells were treated with 10 ng/ml TGF-β3 at indicated time points. Autophagy and target proteins were detected by western blotting in 16HBE cells. (g) 16HBE cells that stably expressed mCherry-EGFP-LC3 fusion protein were treated with TGF-β3. In green and red-merged images, autophagosomes are shown as yellow puncta (i.e.,mCherry⁺EGFP⁺), while autolysosomes are shown as red puncta (i.e.,mCherry⁺EGFP⁻). Autophagic flux was increased when both yellow and red puncta are increased in the cells. Confocal microscopic analysis was shown (2000× magnification). Bar scale, 5 mm. (h) Quantification of the number of LC3 puncta (each group n = 10 images for quantification). Data are representative of three independent experiments and are presented as means ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001, determined by Student's t-test.