Fig. 2

Adipose tissue extracts enable the EMT of CCA cells. Human cholangiocarcinoma (CCA) cell line RBE was incubated with complete growth medium or medium containing human adipose tissue extracts for 24 h. The cell morphology was observed under the invert microscopy (40×) (a). Protein expression of MMP2, MMP9, TGF-β1, claudin 1, occludin, E-cadherin, β-catenin, SNAIL, and Smad3 were analyzed by immunoblot analysis in response to adipose extracts incubation, and the band densitometry analysis was carried out relative to the loading control β-actin or GAPDH (b, c). To determine fatty acid metabolism, the culture medium with different adipose components prior to or after incubation with RBE cells for 24 h were collected for extracellular glycerol and nonestesterified fatty acid (NEFA) detection using colorimetric assays, with the data presented as the absorbance at 490 nm(d, e). The intracellular lipids in adipose cultured RBE cells were visualized by the fluorescence dye Bodipy (20 μg/mL) using confocal microscopy analysis, using Hochest33342 (0.5 μg/mL) for nuclear staining (f). All the samples were prepared in triplicate, and all experiments were repeated at least three times. *P < 0.05