c-Myc enhances transcriptional activation of CLK3 promoter in CCA cells. (A) HEK293T cells were overexpressed with empty vector or Flag–c-Myc with or without CLK3-Luc (−1,328 bp) reporter as indicated. Relative luciferase activity was measured and normalized based on CMV-LacZ expression. (B) Flag–c-Myc or empty vector was transfected into HCCC9810 cells. 24 h later, we performed quantitative RT-PCR and immunoblotting (IB) assays. (C) siRNA against c-Myc or scramble was transfected into HuCCT1 cells. 24 h later, we performed quantitative RT-PCR and immunoblotting assays. (D) Positions of four putative c-Myc–binding motifs (E-box) relative to the transcription start in the CLK3 promoter are indicated. (E) The effect of mutating each of four putative c-Myc–binding sites on the luciferase reporter of CLK3 promoter in HEK293T cells transfected with a c-Myc. Relative luciferase activity was measured. (F) EGF- or TGFβ1-treated HuCCT1 cells were used to perform ChIP with c-Myc antibody. PCR was performed using primers flanking four putative E-box sites of the CLK3 promoter. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, not significant. Data are mean ± SEM and are from three (A, B, right panel; C, lower panel; and E) independent experiments or are representative of three independent experiments with similar results (B, left panel; C, upper panel; and F). P values were calculated using unpaired Student’s t test (A and B) or one-way ANOVA (C and E). TUBA, alpha tubulin.