Fig. 2

TFEB is involved in TGF-β-induced autophagy in PC cells. a Western blot showing expression of LC3B and p62 in MIA PaCa-2 and PANC-1 cells treated with TGF-β (10 ng/mL) for the times indicated. b MIA PaCa-2 and PANC-1 cells transfected with GFP-LC3B were treated with TGF-β (10 ng/mL) for 48 h, and representative confocal images are shown in the left panel; the number of LC3 dots is quantified in the right panel, and at least 30 cells were included in each group. ***, p < 0.001; Scale bar: 20 μm. c Transmission electron microscopy showing formation of autophagosomes/autolysosomes after TGF-β treatment (10 ng/mL, 48 h) in PANC-1 cells. Representative images and their magnified view are shown in the left panel. The right panel is the quantification of the number of autophagic vacuoles from at least 15 areas. Arrowhead: autophagic vacuoles; ***, p < 0.001; Scale bar: 1 μm. d Western blot revealing LC3B and p62 after TGF-β treatment (10 ng/mL, 24 h) in the absence or presence of CQ (10 μM, 24 h) in MIA PaCa-2 and PANC-1 cells. e PANC-1 cells transfected with GFP-mRFP-LC3B were treated with TGF-β (10 ng/mL, 48 h) or CQ (10 μM, 24 h) or (g) pre-transfected with shTFEB followed by a 48-h treatment with TGF-β (10 ng/mL). Representative confocal images of autophagosome (yellow puncta) and autolysosome (red puncta) formation are presented in the upper panel. Scale bar: 20 μm. The numbers of RFP⁺GFP⁺ LC3 puncta and RFP⁺GFP⁻ LC3 puncta are shown in the lower panel. ***, p < 0.001 compared with the control. f Western blot showing LC3B expression in MIA PaCa-2 and PANC-1 cells by transfected with shTFEB and/or treated with TGF-β (10 ng/mL, 48 h)