Fig. 2

CCL3 secreted by adjacent-cancer hepatocytes promotes ICC migration and invasion. a After co-culture with ICC cells, conditioned supernatants secreted by hepatocytes were analyzed for cytokine levels through enzyme-linked immunosorbent assay (ELISA)-based cytokine antibody arrays (Raybiotech). Scan of original blots (left), data analysis schematic (middle), and representative differentially expressed proteins (right) of cytokine antibody arrays (Additional file 3: Fig. S1). b Measurements of levels of cytokine mRNA in THLE3 cells after co-culture with ICC cells were assayed by qRT-PCR. c, d Supernatants were collected and the cytokine secretion level of c CCL23 and d CCL3 were measured in RBE cells through ELISA. e, f Transwell migration assay and matrigel invasion assay e RBE cells and f HuCCT1 cells stimulated with CCL23 (100 ng/ml) or CCL3 (100 ng/ml). Representative images (left) and quantification (right) of Transwell assays are shown. Scale bars, 100 μm. g qRT-PCR and western blot analysis of CCL3 expression in THLE3 hepatocytes following co-culture with ICC cells. h qRT-PCR and western blot analysis of CCL3 expression in RBE cells following the co-culture. i Representative images of IHC staining for CCL3 protein of ICC tumor tissues and their adjacent liver tissues. Red curve: intrahepatic cholangiocarcinoma. Blue arrow, adjacent-cancer hepatocytes; yellow arrow, distant hepatocytes. Scale bars: 100 μm. j Gene ontology (GO) enrichment analysis was performed on the genes that interacted with CCL3. RBE, HuCCT1, human ICC cell lines; THLE3, human hepatocytes cell line. *P < 0.05 **P < 0.01, ***P < 0.001.