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Fig. 1. Jun siRNA suppresses JUN and MMP-2 protein expression in murine microvascular endothelial cells. (A) Growth-quiescent bEND-3 cells transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr or mock-transfected and incubated in medium containing serum for 2 hours before western blot analysis (for JUN or ß-actin) of whole cell extracts. (B) Growth-quiescent bEND-3 cells transfected with 15 µg of pGL3-prom bearing two JUN/AP-1 binding sites and 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr, Egr-1 siRNAscr or mock transfected were incubated in medium containing serum for 24 hours before determination of luciferase activity in the cell lysates. (C) RT-PCR analysis for Jun using extracts of growth-quiescent bEND-3 cells transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr, Egr-1 siRNAscr or mock-transfected and incubated in medium containing serum for 2 hours. (D) RT-PCR analysis for MMP-2 using extracts of growth-quiescent bEND-3 cells transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr, Egr-1 siRNAscr or mock transfected and incubated in serum-free medium containing 10 ng/ml TGFß for 24 hours. (E) Gelatin zymography demonstrating MMP-2 bioactivity in supernatants of transfected bEND-3 cells. The cells were transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA or siRNAscr and incubated in serum-free medium containing 10 ng/ml TGFß for 24 hours. Data are representative of at least two independent experiments performed in duplicate or triplicate and are mean values ± s.e.m. ^*P<0.05 compared with no siRNA control by Student's t-test.

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