Fig. 3. Metabolic requirements for TGF-ß1-induced PAI-1 expression. To assess pathways underlying induced PAI-1 expression, quiescent (Q) T2 cells were stimulated with serum (20%) or TGF-ß1 (1 ng/ml) for 2 hours, in the presence or absence of a 30 minute pretreatment with the indicated inhibitors, prior to RNA isolation (A). Northern blots were hybridized with ³²P-labeled cDNA probes for PAI-1 and A-50 simultaneously. The inability of puromycin to attenuate either serum or TGF-ß1-induced PAI-1 transcripts and the sensitivity of expression to actinomycin D indicated that PAI-1 induction by both stimuli was an immediate-early (i.e., primary) response. TGF-ß1-induced PAI-1 expression in T2 cells is MEK-dependent (B). Quiescent (Q) T2 cells were stimulated with TGF-ß1 (1 ng/ml) for 2 hours in the absence or presence of a 30 minute pretreatment with PD98059 (50 nM) prior to isolation of RNA. Northern blots were hybridized with ³²P-labeled cDNA probes to PAI-1 and A-50.