Contribution of Sp1 to increased expression at 32°C. (A) Effects of Sp1 on endogenous cirp expression. U-2 OS (left) and HEK293 (right) cells were transfected with plasmids expressing Sp1 or shRNA for Sp1 (pSuper-Sp1) as indicated, and one day later transferred to 32°C or remained at 37°C. Six hours after the transfer, cell lysates were analyzed by western blotting using anti-Cirp, anti-Sp1 and anti-actin antibodies. In some experiments, plasmids expressing wild-type Sp1 protein from pSuper-Sp1-resistant mRNA (resist Sp1) were also co-transfected. (−), transfection with empty vector. (+), transfection with the indicated plasmids. Experiments were repeated three (left) or four (right) times, with similar results. (B) Effects on reporter gene expression. HEK293 cells were co-transfected with the F-Luc reporter plasmids driven by the wild-type −500/+56 cirp fragment, pRL-TK, and increasing amounts (+ to +++) of plasmids expressing FLAG-tagged Sp1 (FLAG-Sp1) or pSuper-Sp1 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 the value obtained in cells co-transfected with the reporter, pRL-TK, and empty vector (−). Values are mean ± SE of triplicates, n = 3.