Figure 3.

HSC-T6 conditioned medium can trigger ML1 undergo EMT. A. The morphological change of ML1 after treated with different medium; 1. DMEM with 10% FBS (control); 2. HSC-T6 conditioned medium (CM), 3. Serum free conditioned medium (SF-CM), 4. serum free conditioned medium with 10% FBS (SF-CM+FBS). 5. 10 ng/ml TGF-b. 6. HSC-T6 culturing medium Waymouth. All treatments were added to ML1 cells for 24 h. The group 1-6 represent the same treatment through this figure; B. The immunoblotting result of E-cadherin, N-cadherin, slug, and twist under different conditioned medium to ML1 cells after 12 h treatment; C. The mRNA expression of EMT related transcription factor snail, twist, and slug in ML1 cells with different treatment for 12 h; D. The migration ability of ML1 cells was determined by transwell migration assay at 24 h post treatment, where E shows the quantitative result; F. Immunofluorescence staining of E-cadherin, N-cadherin, α-SMA, vimentin, and Slug in ML1 cells treated for 12 h; G. The cell number was counted by Eosin Y staining after 12-48 h treatment; H. The hepatoma cell line ML1 were treated with HSC-T6 CM for 12-48 h. Cells were fixed with 70% alcohol and stained with 20 μg/ml propidion indium, result showed percentage in each cell cycle stage. Each treatment was triplicated and repeated once with similar result. The expression level was compared to the control group by analyzing band intensity with Image J software.