Fig 4. DHA inhibits TPA-induced uPAR by suppressing the DNA-binding activities of AP-1 in ECV304 cells.

(A) Cells were treated with TPA for 0–90 min, and the cellular extracts were blotted using specific antibodies. (B) Cells were transiently transfected with the pAP-1 luciferase reporter construct. The transfected cells were incubated with TPA for 4 h and the luciferase activities were determined using a luminometer. (C) Cells were treated with 0–20 μM curcumin (Cur) for 1 h prior to exposure to 100 nM TPA for 4 h. After incubation, the uPAR mRNA levels in the cell lysates were determined by RT-PCR. (D) Cells were treated with 0–20 μM curcumin (Cur) for 1 h prior to exposure to 100 nM TPA for 16 h. After incubation, the uPAR protein levels were determined western blotting. (E) The AP-1 decoy oligonucleotide was co-transfected with pGL3-uPAR into cells. After incubation with 100 nM TPA for 4 h, the luciferase activities were determined using a luminometer. (F) Cells were treated with DHA (25, 50, 100 μM) prior exposure to 100 nM TPA, and the expressions of phos-c-fos, phos-c-jun were analyzed by western blotting. (G) Cells were transiently transfected with the pAP-1 luciferase reporter construct, after being pretreated with DHA (25, 50, 100 μM), and then were incubated with 100 nM TPA for 4 h. After incubation, the cells were lysed and luciferase activity was determined. *P<0.05 versus control; **P<0.05 versus only TPA. The data represent the mean ± SD from triplicate measurements.