Induction of MAD1 mRNA and protein expression in response to TGFβ1 in U937 cells. A. Exponentially proliferating U937 promyelocytes were treated with 2.5 ng/ml of TGFβ1. mRNA was purified at the indicated time points. The kinetics of MAD1 expression in response to TGFβ1 were analyzed by quantitative RT-PCR. The mean values ± SD of three independent experiments are shown. B. Whole cell extracts of U937 cells treated as in panel A were generated at the indicated time points and analyzed for endogenous MAD1 protein by western blotting. Tubulin served as loading control. C. U937 cells were pre-treated with the TGFβ receptor I inhibitor SB505124 (5 μM) for 40 min prior to addition of TGFβ1. MAD1 expression was assayed by quantitative RT-PCR 2 hrs after TGFβ1 stimulation. The displayed data are mean values ± SD of three independently performed experiments. D. U937 cells were initially diluted to a density of 105 cells/ml and incubated with 2.5 ng/ml TGFβ1 over a period of 48 hrs. The number of cells was determined at the indicated time-points by CASY measurements.