FIGURE 8.

NFAT proteins mediate the TGF-β switch from growth suppressor to a promoter of cell proliferation. A, relevance of NFAT expression for TGF-β-induced cell proliferation was assessed by [³H]thymidine incorporation assay upon NFAT silencing. PaTu8988t and Panc-1 cells were transfected with either control siRNA or siRNA against NFAT. Cells were then starved and incubated in medium with or without 10 ng/μl TGF-β for 48 h. Bars indicate mean values ± S.D. of three independent experiments. Note that NFAT depletion rendered cells refractory to growth stimulation and partially restored TGF-β growth suppressor activities in PaTu8988T cells. Successful NFAT knockdown was demonstrated by immunoblotting using specific antibodies against NFATc1 and NFATc2 (bottom panel: control siRNA (lane 1), control siRNA + TGF-β (lane 2), siRNA NFAT (lane 3), and siRNA NFAT + TGF-β (lane 4)). B, flow cytometry analysis to study the relevance of NFAT factors in TGF-β induced cell cycle progression of cancer cells. NFAT knockdown cells were treated with TGF-β for 24 or 48 h, respectively, and analyzed by propidium iodide staining and flow cytometry. Cell cycle stages are illustrated in different colors: G2, S, and G1. Loss of NFAT expression restored cell cycle inhibition by TGF-β, as evidenced by increased cells in the G1 phase. Bars indicate mean values ± S.D. of three independent experiments. C, Western blot analysis demonstrating TGF-β regulated cell cycle genes depending on the presence or absence of NFAT expression. Cells were transfected with siRNA against NFATc2 or unspecific control siRNA, serum-starved, and subsequently treated with 10 ng/μl TGF-β for 18, 24, or 48 h. Total cell lysates were then analyzed for expression of NFAT, D-type cyclins and the partnering kinases (CdKs). Protein loading was controlled using anti-β-actin antibodies.