FIGURE 3.

ZNF451 inhibits TGF-β-induced signaling responses. A, stable expression of FLAG-ZNF451 in HaCaT cells. The mRNA level of ZNF451 was detected by using qRT-PCR, and the protein level by Western blotting (inset). Note that the mRNA level of exogenous FLAG-ZNF451 is similar to that of endogenous ZNF451. B and C, ZNF451 inhibits TGF-β-induced reporter gene activity. ZNF451-expressing HaCaT stable cells were transfected with SBE-Luc (B) or PAI-1-Luc (C) reporter. Relative luciferase activity was measured after an 8-h TGF-β treatment (2 ng/ml). Values are the mean ± S.E. of three separate experiments performed in triplicates and normalized for transfection efficiency against Renilla luciferase activities. D–F, ZNF451 blocks TGF-β-induced target gene transcription. ZNF451-expressing HaCaT stable cells were stimulated with 2 ng/ml of TGF-β for 8 h. Total mRNAs were analyzed by qRT-PCR using primers specific to p15 (D), p21 (E), and PAI-1 (F). Values are the mean ± S.E. of three separate experiments performed in triplicates. G, ZNF451 abolishes TGF-β-induced protein expression. ZNF451-expressing HaCaT stable cells (or control cells expressing GFP) were stimulated with 2 ng/ml of TGF-β for 12 h and then harvested for cell lysates. The expression levels of various proteins were examined by immunoblotting with appropriate antibodies as indicated. H, ZNF451 renders cells resistant to TGF-β-induced growth arrest. HaCaT stable cells expressing ZNF451 or control cells expressing GFP were seeded in 96-well plates and treated with or without TGF-β. Cell proliferation was examined using BrdU staining at 2 days, according to the manufacturer's instructions. The number of BrdU-stained cells with TGF-β treatment was scored as the percentage over untreated cells. Values are the mean ± S.E. of three separate experiments performed in triplicates. *, p < 0.05; **, p < 0.01 (Student's t test).