Figure 2

Functional characterisation of the 2G TGFβ reporter cells.

a. U2OS(SEC-C) and 2G TGFβ reporter cells were stimulated with TGFβ for the indicated time points and lysed. The extracts were resolved and immunoblotted with the indicated antibodies. b. U2OS(SEC-C) and 2G TGFβ reporter cells were stimulated with TGFβ for the indicated times and the relative expression of PAI-1 mRNA analysed by qRT-PCR. c. 2G TGFβ reporter cells were left untreated or treated continuously with TGFβ for the indicated time points prior to lysis and luciferase activity measured. d. A pulse of TGFβ treatment for 1h was applied to 2G TGFβ reporter cells and upon TGFβ removal, cells were lysed at the indicated times and the luciferase activity measured. e. 2G TGFβ reporter cells were treated with or without TGFβ and with or without the type I TGFβ receptor inhibitor SB505124. f. 2G TGFβ reporter cells were transfected with siRNAs against non-targeting control (siNT), TGFβR2 or SMAD4. Cells were treated with SB505124 and/or TGFβ as indicated prior to lysis and luciferase activity measured. g. Luciferase activity of 2G TGFβ reporter cell extracts following transfection with vectors encoding the LacI DNA binding domain control or Flag-tagged SMAD3 (1µg per well of 12 well plates). h. 2G TGFβ reporter cells were stimulated with 50pM TGFβ, 50ng/ml EGF or 10 ng/mL PMA for 20h, in the presence or absence of TGFβ pathway inhibitor SB505124, prior to lysis and extracts were subjected to luciferase activity measurements. Bar chart data are represented as mean and error bars indicate standard deviation.