Fig. 4: TACC3 positively regulates the expression of E2F1

a Western blotting revealed that TACC3 knockdown in EJ and 5637 cells caused a decrease in P-AKT, E2F1, and cyclin D1 protein expression. b QPCR revealed that TACC3 knockdown in EJ and 5637 cells repressed E2F1 mRNA expression. Data are presented as mean ± SEM (n = 3). ***p < 0.001 in one-way ANOVA. c QPCR demonstrated that TACC3 overexpression upregulated the level of E2F1 mRNA. Data are presented as mean ± SEM (n = 3). ***p < 0.001 in the Student’s t-test. d EJ or 5637 cells were first transfected with siTACC3 or control siRNA. After 36 h, the cells were co-transfected with the pGL3-E2F1 gene promoter luciferase or the pRL-TK Renilla luciferase construct, and 36 h later, the luciferase activity of the E2F1 promoter was measured and the fold enrichment relative to the control siRNA was determined. Data are presented as mean ± SEM (n = 3). ***p < 0.001 calculated by one-way ANOVA. e Schematic representation of the E2F1 promoter. The region of primers (Supplementary Table 2) used for the ChIP-qPCR assays is indicated. Bar graphs show the fold enrichment relative to IgG. Data are presented as mean ± SEM (n = 3). ***p < 0.001 calculated by the Student’s t-test. Fragment 1(-1258--1077), Fragment 2(-1076--809), Fragment 3(-808--551), Fragment 4(-550--404), Fragment 5(-403--221), and Fragment 6(-220--1). f Cells were first transfected with NC or TACC3 siRNA for 36 h. Then, cells were transfected with the luciferase reporter vector containing the E2F1 promoter or the Δ6 mutant. After 24 h of transfection, the cells were lysed, and the luciferase activity was measured. Data are presented as mean ± SEM (n = 3). p-Values were calculated by one-way ANOVA. **p < 0.01, ***p < 0.001. g, h, Graphs show a positive correlation between TACC3 and E2F1 mRNA levels in bladder cancer cell lines (Pearson’s r = 0.61, p = 0.003) and in bladder cancer clinical tissues (n = 40, Pearson’s r = 0.74, p < 0.001) based on RNA-seq data from a public database