Fig 2: The effect of IFI27 knockdown on VEGF-A in SNU308 cells.Notes: (A) The mRNA expressions of VEGF-A were analyzed in SNU308 IFI27 and SNU308-COLsi cells by RT-qPCR. Data were presented as mean ± SD relative to SNU308-COLsi group (SNU308-COLsi cells group was set as 1). Experiments were performed in triplicate and repeated at least thrice. (B) The amounts of secreted VEGF-A in SNU308 IFI27 and SNU308-COLsi cells culture medium were determined by ELISA. Experiments were performed in triplicate and repeated at least thrice. (C) Luciferase activity of VEGF-A reporter vector-transfected SNU308 IFI27 or SNU308-COLsi cells. Data were presented as mean percentage relative to the control group (SNU308-COLsi group) (± SE, n=6). (D) Western blot showing c-jun and c-fos expressions in SNU308 IFI27 and SNU308-COLsi cells (upper panel) (cropped). Quantitative result of the Western blot (lower panel). Each value was a mean ± SD of three independent determinations relative to the SNU308-COLsi group (SNU308-COLsi group was set as 1). (E) Cell viability of SNU308 cells with or without anti-VEGF-A antibodies treatment for one day as determined by MTT assay. Anti-IgG treatment group was served as the negative control. Data were present as the mean percentage relative to the SU308 cell group (± SD). Experiments were performed in triplicate and repeated at least thrice. (F) Cell viability of HUVECs cultured with mediums from SNU308 IFI27 or SNU308-COLsi cells for one day. Data were presented as mean percentage relative to the SNU308-COLsi group (± SD, n=6) (**P<0.01).Abbreviations: IFI27, interferon α-inducible protein 27; HUVEC, human vascular endothelial cells; SE, standard error; RT-qPCR, real-time quantitative-PCR; VEGF, vascular endothelial growth factor.

Fig 3: Evaluation of IFI27 effect on SNU308 cell growth.Notes: (A) IFI27 mRNA expressions were determined in eight kinds of CCA cells with YSCCC and HUCCT1cells having the lowest and highest IFI27 mRNA expressions, respectively. (B) Knockdown of IFI27 in SNU308 cells. Western blot of IFI27 expression in SNU308 IFI27 and SNU308-COLsi cells (upper panel) (cropped). Quantitative result of Western blot. Each value was a mean ± SD of the three independent determinations relative to the SNU308-COLsi cell group (lower panel) (SNU308-COLsi group was set as 1). (C) Doubling time of SNU308 IFI27 and SNU308-COLsi cells. (D) MTT assay for SNU308 IFI27 and SNU308-COLsi cells at days 1, 2, and 3 were performed. Data were presented as mean percentage relative to the SNU308-COLsi group (± SD, n=6). (E) Histogram of cell cycle distribution (upper panel) and quantitative result of cell cycle distribution (lower panel) of SNU308 IFI27 and SNU308-COLsi cells (*P<0.05, **P<0.01).Abbreviations: CCA, cholangiocarcinoma; IFI27, interferon a-inducible protein 27.

Fig 4: IFI27 knockdown repressed SNU308 cell growth and angiogenesis in vivo.Notes: (A and B) SNU308-COLsi and SNU308-IFI27si cells were xenografted into the nude mice. (C–E) Four weeks later, the tumors were harvested. The volume, weight, and EPO concentration of tumors are measured (*P<0.05, **P<0.01).Abbreviations: EPO, erythropoietin; IFI27, interferon a-inducible protein 27.

Fig 5: The effect of IFI27 knockdown on SNU308 cell metastasis.Notes: (A) The migration ability of SNU308 IFI27 and SNU308-COLsi cells. Data were presented as mean percentage relative to the SNU308-COLsi group (± SD). Experiments were performed in triplicate and repeated at least thrice. (B) The invasion ability of SNU308 IFI27 and SNU308-COLsi cells. Data were presented as mean percentage relative to the SNU308-COLsi group (± SD). Experiments were performed in triplicate and repeated at least thrice. (C) The Western blot of E-cadherin, N-cadherin, and fibronectin expressions in SNU308 IFI27 and SNU308-COLsi cells (upper panel) (cropped). Quantitative result of the Western blot (lower panel). Each value was a mean ± SD of three independent determinations relative to the SNU308-COLsi group (SNU308-COLsi group was set as 1). (D) Immunofluorescence staining of F-actin (green) expression of SNU308 IFI27 and SNU308-COLsi cells. DAPI (red) was applied for nucleus stain (*P<0.05, **P<0.01).Abbreviations: F-actin, filamentous actin; IFI27, interferon a-inducible protein 27; P, propidium iodine.