Fig. 2

Effects of 2-month BBN treatment on RhoGDIβ mRNA 3’-UTR activity and miR-219a expression in UROtsa cells. A UROtsa cells were treated with 400 µM BBN for 3 days, 2 months, and 6 months. The cells then had their total RNA extracted and then processed for measures of RhoGDIβ mRNA levels using real-time PCR. B UROtsa^C2mo and UROtsa^BBN2mo cells were pre-treated with 5 µM MG132 for 6 h, the cells were then subjected to determining RhoGDIβ protein degradation rates in the presence of CHX (50µg/mL) for different lengths of time. The bands of RhoGDIβ and β-actin were not derived from the same membrane due to big differential binding intensities of two antibodies. C Indicated cell extracts were evaluated for levels of S6 phosphorylation at Ser235/236 and total S6 using Western blot. GAPDH was used as a protein loading control. D RhoGDIβ 3’-UTR-driven luciferase and pRL-TK reporters were transiently co-transfected into UROtsa^C2mo and UROtsa^BBN2mo cells. The luciferase activity of each transfectant was then evaluated. Bars represent mean ± SD from three independent experiments. The asterisk indicates significant change compared with control transfectant (p < 0.05). E Potential miRNA binding sites in RhoGDIβ mRNA 3’-UTR were predicted using the TargetScan and miRcode databases. F Quantitative real-time PCR was used to measure miRNA expression. Bars represent mean ± SD from three independent experiments. The asterisk indicates significant increase in comparison to vehicle control cells (p < 0.05)