FIG 3.

FHL2 is a negative regulator of the TGF-β1 promoter. (A) Luciferase reporter assay in WT and Fhl2^−/− MEFs. The activity of the −800/+800 reporter in WT MEFs was arbitrarily set at 1. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n.s., nonsignificant. (B) Analysis of FHL2 gene transcription by qPCR. Primers to detect murine Fhl2 were used in WT and Fhl2^−/− MEFs. To restore FHL2 expression in Fhl2^−/− MEFs, human FHL2 was stably expressed in Fhl2^−/− MEFs (FHL2-r). Transient transfection of 0.2 μg or 0.8 μg of human FHL2 was carried out in FHL2-r cells. Human FHL2 primers were used to analyze mRNA in FHL2-r-related cells. (C) Analysis of FHL2 protein in MEFs and 293T cells with anti-FHL2 antibody reactive to both human and mouse FHL2 proteins. (D) The TGF-β1 promoter is negatively regulated by FHL2. The −800/+510 reporter was transfected into MEFs expressing different doses of FHL2. The cells were treated with 10 μM SB431542 to inhibit the effects of endogenous TGF-β, followed by induction for 1 h with human TGF-β1 (2 ng/ml), before being subjected to luciferase assay. (E) Reporter assay in 293T cells. 293T cells were cotransfected with 0.2 μg or 0.8 μg of plasmid expressing FHL2 and the murine TGF-β1 −800/+510 promoter reporter; 36 h later, the cells were treated with 10 μM SB431542, followed by induction for 1 h with human TGF-β1, before being subjected to luciferase assay. The data presented are the means and SD obtained from three independent experiments.