Involvement of Sp1 in RA induction of uPA. (A) Promoter assay. Transfection study was performed as described before using wild-type pUK-luciferase (pUK-Luc, samples 1 through 3), pUK GC box-luciferase (pUK GC-Luc, samples 4 through 6), and pUK-luciferase deficient in GC box (pUK ▵GC-Luc, samples 7 through 9). Data are expressed as the relative luciferase activity compared with the activity of pUK-Luc cotransfected with empty pSG5 and untreated with 9cRA (sample 1). Samples 1, 4, and 7, reporter only; samples 2, 5, and 8, RAR and RXR; samples 3, 6, and 9, RAR and RXR + 9cRA. (B) Transcription factor decoy experiments. After BAECs were transfected with various amounts of Sp1 decoy or its mutant, whose sequences are presented above the illustration, the cultures were incubated either with vehicle or with 1 μmol/L atRA for 12 hours, cell lysates were prepared, and cellular PA levels were determined. Curves a and b, RA-treated cells; curves c and d, unstimulated cells. Curves a and d, mutant oligo; curves b and c, Sp1 decoy. (C) Cellular PA levels were determined after treatment of BAEC cultures with various concentrations of atRA for 12 hours in the absence (curve a) or presence (curve b) of 10 nmol/L mithramycin. For (A) through (C), the results are presented by the average ± SD (n = 3). Each similar experiment was repeated three times and representative results are shown here. (D) After treatment of BAECs with 1 μmol/L atRA for 12 hours in the absence (lane 1) or presence (lane 2) of 10 nmol/L mithramycin, nuclear extracts were prepared and Sp1 binding to uPA GC box was determined by gel shift assays as described in Materials and Methods.