Fig 2: CCL20 and CCR6 are significantly increased in HCC tissues and cell lines. (A) RT-qPCR analysis of DEPDC1, CCL20 and CCR6 mRNA in HCC tissue and matched adjacent normal liver tissue. (B) Analysis of RT-qPCR results with significant Pearson correlation analysis of DEPDC1 with CCL20 and DEPDC1 with CCR6 in HCC. (C and D) Immunohistochemistry assays were used to determine the protein expression of DEPDC1, CCL20 and CCR6. Scale bar, 25 µm. (E) The correlation of DEPDC1 with CCL20 and CCR6 was determined via Pearson's correlation analysis. (F) Western blotting was performed to determine the protein expression of CCL20 and CCR6 in four human HCC cell lines (Li-7, Huh-7, SNU-387 and Hep3B) and one normal human hepatic cell line (L02). *P<0.05, **P<0.01. CCL20, chemokine (C-C motif) ligand 20; CCR6, chemokine (C-C motif) receptor 6; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR; DEPDC1, DEP domain containing 1.

Fig 3: Schematic model depicting the mechanism of DEPDC1-induced HCC progression. DEPDC1 promotes HCC cell proliferation, colony formation and invasion via the CCL20/CCR6 axis. DEPDC1 also induces the activation of p-Akt via the CCL20/CCR6 axis, thereby promoting the protein expression of c-myc and cyclin E1 and HCC cell proliferation. DEPDC1-induced HCC cell-derived CCL20 regulates HUVEC angiogenesis and invasion by mediating endothelial CCR6. DEPDC1, DEP domain containing 1; HCC, hepatocellular carcinoma; CCL20, chemokine (C-C motif) ligand 20; CCR6, chemokine (C-C motif) receptor 6; p-, phosphorylated; HUVEC, human umbilical vein endothelial cells.

Fig 4: CCL20 or CCR6 knockdown partially reverses the effect of DEPDC1 overexpression on HCC cell proliferation, colony formation and invasion in vitro. Li-7 and Hep3B cells were treated with a combination of pcDNA3.1 and NC plasmids or a combination of DEPDC1 expression vectors with NC, CCL20 siRNA or CCR6 siRNA. (A) Cell Counting Kit-8 and (B) EdU assays were performed to determine cell proliferation. Scale bar, 75 µm. (C) Foci formation assays were utilized to observe colony formation. (D) Cell invasion was determined via a Transwell assay. Scale bar, 50 µm. (E) Western blotting was performed to detect the protein expression of p-Akt, Akt, c-myc and cyclin E. Each experiment was performed in triplicate. *P<0.05, **P<0.01. CCL20, chemokine (C-C motif) ligand 20; CCR6, chemokine (C-C motif) receptor 6; DEPDC1, DEP domain containing 1; HCC, hepatocellular carcinoma; NC, negative control; siRNA, small interfering RNA; p-, phosphorylated.