Fig. 5
c-Jun phosphorylation mediated RhoGDIβ mRNA transcription in UROtsa cells treated with BBN for 6 months. A UROtsaC6mo and UROtsaBBN6mo cells transiently transfected with RhoGDIβ promoter-driven luciferase reporter were used to determine RhoGDIβ promoter transcriptional activity. Results are presented as RhoGDIβ promoter activity relative to vehicle control (relative RhoGDIβ promoter activity). Bars represent mean ± SD from three independent experiments. The asterisk (*) indicates a significant increase from vehicle control (p < 0.05). B Schematic of putative transcription factor consensus binding sites in the RhoGDIβ proximal promoter region predicted using bioinformatics analysis. C Expressions of potential transcription factors were determined by Western blot in UROtsa cells following BBN (400 μM) exposure for the indicated periods. β-Actin was used as a protein loading control. D, E TAM67 was stably transfected into UROtsaBBN6mo cells, and the stable transfectants were then evaluated for c-JUN (D) (TAM67) and RhoGDIβ expression by Western blot (D) and RhoGDIβ promoter activity (E). F, G Schematic of two different c-Jun point mutations in RhoGDIβ promoter-driven luciferase reporter. H Wild-type RhoGDIβ promoter-driven luciferase reporter or its mutants at the c-Jun binding site were co-transfected with pRL-TK into UROtsaC6mo and UROtsaBBN6mo cells. Luciferase activity of each transfectant was evaluated, and the results were presented as relative RhoGDIβ promoter activity. The asterisk indicates significant increase from control group (p < 0.05). The number sign indicates significant decrease between WT and c-Jun-1 Mut in UROtsaBBN6mo cells (p < 0.05). I ChIP assay using anti-c-Jun antibody to detect the interaction between c-Jun and the RhoGDIβ promoter