Analysis of PAI-1 reporter gene activity and protein expression upon coexpression of full-length and dominant-negative Nur77 constructs.

(A) PAI-1 reporter gene activity. Bars 1 to 3 represent overexpression of Nur77 as well as TNFα induction in HUVECs; bars 4 and 5 represent overexpression of a dominant-negative mutant of Nur77 lacking its transactivation domain in untreated or TNFα-stimulated HUVECs; bars 6 and 7 represent the activity of a PAI-1 reporter gene construct lacking the 20 bp containing the NBRE with and without TNFα stimulation. Black bars are TNFα⁺(±SD of triplicates). (B) 4 × NBRE reporter gene activity. Effect of TNFα stimulation and overexpression of Nur77 on activity of a reporter gene construct containing a 4 × tandem repeat of an NBRE fused to a minimal promoter. Black bars are TNFα⁺ (±SD of triplicates). (C) Immunocytochemistry. HUVECs were transiently transfected to express EGFP or dnNur77-EGFP. After treatment with TNFα for 4 hours, cells expressing EGFP alone (green nuclear and cytoplasmatic signal) as well as untransfected cells produced large amounts of PAI-1 protein (red), whereas cells expressing dnNur77 (green, nuclear localization) showed no expression of PAI-1. Original magnification × 100 (C).