Induction of uPA and RARs by RA in endothelial cells. (A) Cell lysates were prepared from confluent BAEC cultures after the cultures had been incubated in MEM-BSA with 1 μmol/L atRA for varying lengths of time. Total RNA was isolated from cell lysates, and the changes in uPA mRNA levels as well as RARs and RXRs mRNA levels were assessed by Northern blotting as described in Materials and Methods. (B) After BAECs were treated for the indicated times with 1 μmol/L atRA in the absence or presence of 6 μg/mL cycloheximide (CHX), changes in uPA mRNA levels were determined by Northern blotting. (C) After exposure to either vehicle or 1 μmol/L atRA for 12 hours, BAECs were treated for the indicated times with 1 μg/mL actinomycin D. The rate of disappearance of uPA mRNA was determined by Northern blotting. Because mRNA for GAPDH also decreased along with incubation with actinomycin D, we presented ethidium bromide-labeled 28S RNA as internal. (D) Northern analyses were performed using total RNA isolated from human aortic endothelial cells treated with 1 μmol/L atRA for 12 hours.