Fig. 5. Metabolic requirements for CD-induced PAI-1 mRNA expression. Serum-deprived cells were pretreated for 30 minutes with actinomycin D (Act D; 5 µg/ml) or puromycin (Puro; 100 µg/ml) prior to a 4 hour exposure to CD (final concentration 10 µM). Optimal concentrations of inhibitors were determined previously (e.g. Ryan et al., 1996; Hawks and Higgins, 1998). Northern blots were probed with ³²P-labeled cDNAs to PAI-1 and A50 (A). CD-induced PAI-1 transcription appeared to utilize a secondary (i.e. protein-synthesis-dependent) response mechanism. To evaluate the potential role of autocrine TGF-ß as a secondary response intermediate, quiescent R22 cell cultures were pretreated for 30 minutes with TGF-ß neutralizing antibodies prior to addition of either CD or TGF-ß1. RNA was isolated, and blots were probed for PAI-1 and A50 transcripts (B). Representative blots are shown.