Fig. 4

Fatty acid binding protein 4 (FABP4) is involved in the metastasis of CCA. Human CCA cell RBE was treated with or without human adipose tissue extracts for 24 h. The mRNA levels of FABP 1 to 7 were determined using qRT-PCR with β-actin as control gene (a). To detect the protein expression of FABP4, 50 μg of human adipose tissues, undifferentiated or fully differentiated pre-adipocyte 3 T3-L1, as well as CCA cell RBE and Hccc-9810 were analyzed by Western blot assay, using GAPDH as loading control (b). To measure CCA tissues contained lipids, patient CCA tissues (n = 3) were freshly made into 5 μm frozen sections, stained using OilRed solution, and observed under the microscopy (40×) (c). To monitor the uptake of exogenous FABP4 protein by CCA cells, exponential monolayer RBE was incubated with synthesized EGFP-tagged FABP4 for 2 h or 4 h, using EGFP as a control. The intracellular distribution of FABP4 was visualized using the living cell imaging system (d). All the samples were prepared in triplicate, and all experiments were repeated at least three times. *P < 0.05