Reporter construct gene expression analysis.

<p>(A) The <i>UBC</i> promoter-pGL3 constructs P1 (-916/+878) and P3 (-371/+878) containing FR1-2-6 and only FR6, respectively, were transfected in HeLa cells. Fourty-eight hours post-transfection, cells were treated with the DMSO vehicle or MG132 (20 μM). After 8h, luciferase mRNA levels were determined by RealTime PCR. Expression data, normalized to the housekeeping GAPDH gene, were analyzed by the 2^-ΔΔCT method and referred to the value obtained in DMSO-treated cells. The data represent the mean fold induction ± S.E. of 5 independent experiments. Asterisks indicate statistical significance versus cells receiving only the vehicle (*p<0.05; **p<0.01). Statistical analysis was performed on ΔCT values with one-way ANOVA. (B) The wild-type <i>UBC</i> promoter-pGL3 construct P1 (FR1-2-6) and its mutant counterparts P1mut FR1-2 and P1mut FR6, where HSF binding motifs were selectively mutated within FR1 and FR2 altogether or FR6, respectively, were transfected in HeLa cells. Forty-eight hours post-transfection, cells were treated with the DMSO vehicle or MG132 (20 μM). After 8h, luciferase mRNA levels were determined by RealTime PCR. Expression data, normalized to the housekeeping GAPDH gene, were analyzed by the 2^-ΔΔCT method and referred to the value obtained in DMSO-treated cells. The data represent the mean fold induction ± S.E. of 7 independent experiments. Asterisks indicate significant differences of the MG132-induced LUC mRNA fraction between cells transfected with wild-type and mutant reporter constructs (*p<0.05; **p<0.01). Statistical analysis was performed on ΔΔCT values with one-way ANOVA.