Figure 5.

Dual luciferase reporter assay. CDC6 short and long 3′-UTRs were cloned into the 3′-UTR of Firefly luciferase gene in pMIR. pMIR–short, pMIR–long and pMIR–empty vectors were co-transfected with phRL–TK into MCF7 cells. MCF7 cells were kept in phenol red-free medium supplemented with 10% charcoal-stripped FBS for 48 h. Twelve hours post-transfection, E2 was applied to a final concentration of 10 nM for 12 h. E2 responsive TFF1-promoter construct was used as a positive control for E2 treatment. pGL3 is the empty vector, TFF1-ERE (in pGL3) harbors the promoter and E2-responsive regions of the TFF1 gene. Dual-luciferase assay was performed 24 h after transfection. Firefly/Renilla luciferase read-outs from the constructs were normalized to that of empty pMIR, which was set to 1. ***Indicates P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test).