FIG. 4.

VEGF activates AP-1- and NFAT-dependent transcription- (A) HUVECs were transfected by the calcium phosphate procedure with the AP-1-responsive (−73 to +63) region of the collagenase promoter plasmid for 4.5 h and stimulated or left untreated for an additional 16 h, and then the luciferase activity was determined. The data is expressed as the mean and standard deviation (error bars) of determinations performed in triplicate. Results of a representative experiment are shown. Four different experiments yielded similar results. (B) HUVECs were transfected with an NFAT-dependent luciferase reporter plasmid. At 16 h after transfection, the cells were pretreated or not (−) with CsA (200 ng/ml) for 2 h, and then left untreated or further stimulated for an additional 6 h with VEGF or a combination of PMA and calcium ionophore (P/I₀). The results are expressed as the relative fold induction over the relative luciferase units (RLU) displayed by the corresponding transfected unstimulated cells. Results of a representative experiment of five are shown. VEGF (50 ng/ml), PMA (20 ng/ml), and A23187 calcium ionophore (1 μM) were used at the same doses in single or combined treatments in panels A and B.