CLK3 in CCA is significantly up-regulated and associated with decreased OS and acts as an oncogene. (A) Examining the mRNA (upper panel) and protein (lower panel) levels of CLK3 in normal cholangiocytes and CCA cell lines. (Bi) Serial slides were incubated with either mouse IgG or CLK3 antibody for IHC detection. Bar, 100 µm. (Bii) IHC analysis of CLK3 expression in a CCA patient’s tissue array (n = 100). Representative pictures are presented. Bars, 100 µm. (C) Stratified by CLK3 levels in CCA patients (n = 84), Kaplan-Meier analysis of OS is shown. (D and Ei) MTT assays were performed to compare the proliferation of HCCC9810 cells with Dox-induced CLK3 expression or without, which was confirmed using immunoblotting. (Eii) BrdU assay in D. (Eiii) Soft agar assays in D. Bar, 100 µm. (Fi and Fii) Transwell assays and wound healing using HCCC9810 treated as in D. (Gi) The effect of CLK3 overexpression induced by Dox on HCCC9810 xenografts. (Gii and Giii) Dox-inducible expression of CLK3 in HCCC9810 cells significantly promoted the number of CCA abdominal metastatic nodules (ii) and lung metastatic nodules (iii) compared with their controls. (Hi and Hii) MTT assays were performed to compare the proliferation of Sk-hep1 and HCT116 cells with CLK3 knockdown, which was confirmed using immunoblotting. (Hiii) Transwell assay in Hi. (I) MTT assays and wound healing using HiBEC treated as in D. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, not significant. Data are mean ± SEM and are from three (F, G, Hii, Hiii, and I) and four (E) independent experiments or representative of three independent experiments with similar results (A lower panel, D, and Hi). P values were calculated using unpaired Student’s t test (E–G, Hii, Hiii, and I) or one-way ANOVA (A) or χ-square test (Bii). KD, knockdown; TUBA, alpha tubulin.