LPS induction of TF expression in THP-1 cells is inhibited by PD98059.

(A) THP-1 cells were preincubated with various doses of PD98059 (0.1, 0.5, 5, 10, 20 μM) prior to stimulation with LPS (10 μg/mL) for 5 hours. TF activity was determined in triplicate using a one-step clotting assay. Data (mean ± SD) from 3 independent experiments are shown. (B) TF antigen was detected by Western blotting in LPS-stimulated (5 hours) THP-1 cells with or without preincubation with PD98059 (25 μM). (C) Total RNA was extracted from THP-1 cells stimulated with LPS for 2 or 4 hours at 37°C after preincubation for 30 minutes with or without PD98059 (50 μM). TF mRNA levels were determined by Northern blotting. The membrane was reprobed with a G3PDH probe to assess RNA loading. (D) The pTF-LUC (3 μg) was transiently transfected into THP-1 cells. After 46 hours, transfected cells were preincubated in the presence or absence of PD98059 (25 μM) for 30 minutes prior to incubation with or without LPS for 5 hours at 37°C. Luciferase activity in cell lysates was determined, and results were expressed as fold induction. Data (mean ± SD) are from 3 independent experiments.