Tacrine hydrochloride inhibits CCA with aberrant CLK3 expression. (A) Schematic representation of screening CLK3 inhibitors using the US drug collection. pEGFP-CLK3-Q607R mutant was stably transfected into HCCC9810 cells, which then were treated with 1,280 drugs. The compounds were screened based on change in fluorescence intensities. (B) 20 candidate drugs are shown by heatmap of CLK3 levels. (C) HCCC9810 and HuH-28 cells stably expressing CLK3-Q607R or empty vector were treated with tacrine hydrochloride for 24 h. Immunoblotting was performed as indicated. (D) Measuring the levels of purine in HCCC9810 and HuH-28 cells treated as indicated. (E and F) The effect of tacrine hydrochloride on the aggressiveness of HCCC9810 cells stably expressing CLK3-Q607R mutant. (G) The effect of tacrine hydrochloride on CCA growth in mice treated as indicated. *, P < 0.05; **, P < 0.01. Data are representative of three independent experiments with similar results (B and C) or are from three independent experiments (D–G; mean ± SEM). P values were calculated using unpaired Student’s t test (E and G) or one-way ANOVA (D and F). TUBA, alpha tubulin.