Figure 6.

HuR levels and cisplatin treatment affect the activity of a reporter construct bearing the DNMT3b 3′UTR. Two days after siRNA transfections, the expression vector pMIR-report (firefly luciferase reporter system) containing either DNMT3b 3′UTR (pMIR-3′UTR) or GAPDH 3′UTR (pMIR-GAPDH) were transiently cotransfected into RKO cells along with pGL4-Renilla (used to normalize for transfection efficiency); 24 h later, cells were treated with cisplatin (50 μM, 8 h), and protein and RNA were collected. (A) Protein extracts were used for the detection of firefly and renilla luciferase activities. Graph shows the relative fold increase in firefly luciferase activity seen in the pMIR-DNMT3b transfection group relative to pMIR-GAPDH after normalization to renilla activity. Values represent the means ± SEM from three independent experiments (*P ≤ 0.05). (B) Reverse-transcribed RNA was used for qPCR detection of the reporter DNMT3b 3′UTR-luciferase mRNA relative to the GAPDH 3′UTR-luciferase mRNA. Transfection efficiency was normalized to the amount of pMIR vector present in each sample, which was quantified by qPCR amplification of the CMV promoter region. Data represent the means ± SEM from three independent experiments.