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Figure 1: Blockade of autocrine TGF-ß signaling in murine breast cancer NMuMG-ST cells by the expression of a dominant-negative RII (DNRII). (A) NMuMG-ST Control and DNRII cells were treated with or without TGF-ß3 (0.5 ng/ml) for 24 hours. The expression of endogenous TGFß RII receptor (TßRII), DNRII and p-Smad3 were detected by Western blot analysis. (B) Control and DNRII cells were transiently co-transfected with a TGF-ß responsive promoterluciferase construct, pSBE4-Luc, and a ß-galactosidase expression construct. The transfected cells were treated with or without TGF-ß3 (0.5 ng/ml). The activity of luciferase and ß-galactosidase in the cell lysates were measured after 24 hours. ß-galactosidase was used to normalize the luciferase activity and the data represent the means + SEM from triplicate transfections (*P<0.05). (C) The cells were plated in a 96-well plate and treated with or without TGF-ß3 (0.5 ng/ml) to determine if the cells were sensitive to TGF-ß-mediated growth inhibition. At various time points, MTT reagent was added to each well for 2 hours and aspirated. DMSO was added and absorbance at 595 nm in each well was obtained with a microplate reader. Each data point is mean+SEM from 4 replicate wells. |