Fig. 8: c-Myc transcriptionally activates TPI1 to promote Gem resistance in BCa.

A Identification of c-Myc as a potential transcription factor regulating TPI1 using four TF prediction network tools and correlation analysis in the TCGA-BLCA dataset. B, C c-Myc knockdown models in J82 and UMUC3 cells. Knockdown efficiency was confirmed by Western blot. D Western blot analysis of TPI1 protein expression in c-Myc knockdown cells. E, F Gem sensitivity assays in c-Myc knockdown J82 and UMUC3 cells. G, H c-Myc overexpression models in J82 and UMUC3 cells. Overexpression efficiency was confirmed by Western blot. I Western blot analysis of TPI1 protein expression in c-Myc-overexpressing cells. J Prediction of three potential c-Myc binding sites in the TPI1 promoter using the JASPAR database. K Dual-luciferase reporter assay analyzing the regulation of TPI1 promoter activity by c-Myc using reporter plasmids containing either the wild-type TPI1 promoter (TPI1-p-WT) or its binding site-mutated version (TPI1-p-MT). Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis assessing the binding of endogenous c-Myc protein to the TPI1 promoter region in J82 (L) and UMUC3 (M) cells. N, O TPI1 knockdown in c-Myc-overexpressing J82 cells. Knockdown efficiency was confirmed by Western blot and quantification. P, Q Colony formation assays show that TPI1 knockdown rescues the c-Myc overexpression-induced promotion of cell proliferation and quantification. R TPI1 knockdown reverses the Gem resistance caused by c-Myc overexpression. Data are presented as mean ± SD from three independent experiments. P values in (C, D, O, Q) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. P values in (E, F, H, I, K, L, M, R) were calculated using a two-tailed unpaired Student’s t test.