PMC:539061 / 35235-37335 JSONTXT

{"project":"2_test","denotations":[{"id":"15630473-12646922-84810285","span":{"begin":409,"end":410},"obj":"12646922"},{"id":"T81547","span":{"begin":409,"end":410},"obj":"12646922"}],"text":"TGF-β2, but Not Wnt/noggin, Transiently Induces Snail Expression In Vitro\n(A) Failure of Wnt and noggin signaling to induce Snail in cultured keratinocytes. Primary mouse keratinocytes were treated with Wnt- and/or noggin-conditioned medium (+) or the corresponding control medium (–). These conditions are known to activate the LEF-1/β-catenin reporter TOPGal and down-regulate the E-cadherin promoter (see [4] for details). Using Western blot analyses, cellular proteins were then analyzed for Snail, LEF-1, β-catenin, and tubulin. Proteins from keratinocytes transfected with K14-Snail were used as a positive control for Snail expression.\n(B) TGF-β2 can induce Snail protein. Primary keratinocytes were treated for the indicated times with recombinant TGF-β2 (+) or heat inactivated TGF-β2 (–).Total cellular proteins were then isolated and analyzed by Western blot for Snail, pSMAD2 (reflective of activated TGF- signaling), and tubulin. Note the activation of Snail expression, peaking at 2 h post-TGF-β2 treatment and then disappearing thereafter.\n(C) Snail mRNA expression is transiently induced by TGF-β2. The experiment in (B) was repeated, and this time, total RNAs were isolated from keratinocytes treated with TGF-β2 for the indicated times. RT-PCR was then used with (+) or without (–) reverse transcriptase (RT) and with primer sets specific for Snail and GAPDH mRNAs. Note that Snail mRNA expression also peaked at 2 h, paralleling Snail protein.\n(D) TGF-β2 treatment results in enhanced activity of a Snail promoter-β-galactosidase reporter. Keratinocytes were transfected with a β-galactosidase reporter driven by a Snail promoter that is either WT (wt prom) or harbors a mutation in a putative pSMAD2/pSMAD4 binding site (mt prom). At 2 d posttransfection, cells were treated with either TGF-β or heat-inactivated TGF-β2 (inact) for the times indicated. β-galactosidase assays were then conducted, and results are reported as fold increase over a basal level of activity of 1. The experiment was repeated three times in triplicate, and error bars reflect variations in the results."}