Fig. 6

Stimulation of PAI-1 expression in 3T3-L1 adipocytes by TGF-β.

3T3-L1 adipocytes were grown and differentiated in 6-well tissue culture plates as described previously (28). For kinetic studies (A), total RNA was isolated from untreated cells (●), and cells treated with 1 ng/ml TGF-β (■) for the indicated times, and the steady-state levels of PAI-1 mRNA were determined using quantitative PCR. Inset: Conditioned medium (20 µl) from untreated cells and cells treated with TGF-β for various times were electrophoresed under nonreducing conditions on a 9% SDS-PAGE gel and the proteins were transferred to nitrocellulose membranes. The membranes were analyzed for PAI-1 antigen by Western blotting using a polyclonal rabbit anti-mouse PAI-1 antiserum and the enhanced chemiluminescence detection system. For dose-response experiments (B), total RNA was isolated from untreated cells, and cells treated with increasing amounts of TGF-β for 3 hr. Steady-state levels of PAI-1 mRNA were determined as above. Inset: Western blot analysis for PAI-1 antigen in conditioned medium (20 µl) from untreated cells, and cells treated with increasing amounts of TGF-β for 24 hr. For both A and B, n = 6 ± SD.