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. 2020 Nov 3;47(1):256–266. doi: 10.3892/ijmm.2020.4778

Figure 2.

Figure 2

TGF-β1 simultaneously induces activation and autophagy in JS1 cells. (A) On day 5 after isolation of primary HSCs, the cells were fixed, and lipid droplets were stained using BODIPY581/591 after treatment with 10 ng/ml TGF-β1 for 24 h. The quantity of droplets per cells was quantified. Scale bar, 20 µm. *P<0.05 vs. control. (B) JS1 cells were treated with TGF-β1 (5 or 10 ng/ml) for 24 h. Western blotting was performed to assess the expression of fibrosis-associated markers α-SMA and Col.I. (C) mRNA expression levels of α-SMA and Col.I in JS1 cells were determined using reverse transcription- quantitative PCR following treatment with 10 ng/ml TGF-β1 for 24 h. (D) JS1 cells were either treated with 10 ng/ml TGF-β1 either alone or in combination with 100 ng/ml rapamycin for 2 h. Cells were fixed and subsequently imaged using transmission electron microscopy. Autophagosomes (indicated by the white arrows) were clearly visible in the cytoplasm and were more abundant in cells treated with both TGF-β1 and rapamycin. Scale bar, 2 µm. (E) Protein expression levels of LC3BII and cleaved-caspase-3 were detected by western blotting after incubation of JS1 cells with 10 ng/ml TGF-β1 for 2, 4, 8, 12 or 24 h. (F) mRNA expression levels of Beclin-1, Atg5, p21, Bcl-2 and p53 in JS1 cells incubated with 10 ng/ml TGF-β1 for 24 h. (G and H) JS1 cells were transfected with an RFP-GFP-LC3B virus vectors for 24 h, and subsequently treated with 10 ng/ml TGF-β1 alone or in combination with 50 µM CQ for 24 h. Cells were imaged using a confocal microscope. A total of 10 different fields (10 cells per field) were randomly selected and manually counted. The percentage of cells with yellow (colocalization of GFP and RFP) and red puncta (autophagosomes and lysosomes merged with GFP quenched) were compared with the control and treatment with CQ groups alone. TGF-β1 significantly increased the number of autophagosomes and autolysosomes. Scale bar, 20 µm. **P<0.01 vs. control; &&P<0.01 vs. CQ. (I) JS1 cells were incubated with 10 ng/ml TGF-β1 for 12 h, and subsequently pretreated with 5 mM 3-MA, 50 µm CQ for 2 h, or 100 ng/ml rapamycin for 2 h. LC3BII was detected using western blotting. Protein ratios were used to quantify fold change relative to the control. The mRNA expression levels were expressed as the fold-change relative to control. *P<0.05 and **P<0.01 vs. control; #P<0.05 and ##P<0.01 vs. TGF-β1. Data are presented as the mean ± the standard error of the mean. LC3B, microtubule-associated protein 1 light chain 3B; α-SMA, α-smooth muscle actin; HSC, hepatic stellate cell; Col.I, collagen type I; TGF-β1, transforming growth factor-β1; CQ, chloroquine; 3-MA, 3-methyladenine; C-Casp3, cleaved caspase-3; Rapa, rapamycin.