Silencing CLK3 strongly suppresses the aggressiveness of CCA cells. (A and B) MTT assays for measuring the proliferation of the HuCCT1 and RBE cells with or without 4 µg/ml Dox-induced knockdown of CLK3, which was confirmed using immunoblotting. (C) BrdU incorporation assay was performed using samples in A. Bars, 100 µm. (D) Examining anchorage-independent growth using samples in A. Bars, 100 µm. (E and F) Wound healing and transwell experiments using samples in A. Bars, 100 µm. (G) The effect of CLK3 deficiency induced by Dox in HuCCT1 and RBE cells on mice xenografts. Bars, 1 cm. (H) Dox-inducible knockdown of CLK3 in HuCCT1 and RBE cells significantly inhibited the number of CCA abdominal metastatic nodules. Arrows indicate metastatic foci. *, P < 0.05; **, P < 0.01; ***, P< 0.001. Data are mean ± SEM and are from three (B and E–H), four (C), and five (D) independent experiments or are representative of three independent experiments with similar results (A). P values were calculated using unpaired Student’s t test (B and G) or one-way ANOVA (C–F and H). KD, knockdown; TUBA, alpha tubulin.