Effects of MCRE mutation on Sp1 function. (A) HEK293 cells were co-transfected with the F-Luc reporter plasmids driven by the −500/+56 cirp fragment containing wild-type or mutant MCRE, pRL-TK, and plasmids expressing FLAG-tagged Sp1 (FLAG-Sp1, A) or shRNA for Sp1 (pSuper-Sp1, B) as indicated. One day after transfection, cells were transferred to 32°C or remained at 37°C. Twenty-four hours later, luciferase activities were analyzed. F-Luc activity was normalized to R-Luc activity and expressed as relative to that obtained with wild-type MCRE and without modulation of Sp1 level (−). Values are mean ± SE of triplicates. *, significantly different (p <0.05). Experiments were repeated three times, with similar results.