FIG. 4.

Transfection of 5-LOX and FLAP expression vectors restored ROI production and ROI-dependent NF-κB activity in MCF7 A/Z cells. (A) MCF A/Z cells were transfected with the HIV-κB-CAT reporter plasmid alone or together with a FLAP expression vector, and CAT activities were measured in unstimulated cells or in cells stimulated with IL-1β for 6 h (50 U/ml), as indicated in the figure. The IL-1β treatment was performed in the absence or in the presence of either the FLAP inhibitor MK886 at 0.5 μM (column 5) and 1 μM (column 6) or the 5-LOX inhibitor ETYA at 35 μM (column 8) and 65 μM (column 9). Each column represents the mean of three independent experiments (± SD). We did not observe any statistically significant differences between cells stimulated by IL-1β in the presence or absence of the inhibitors. The total amount of transfected DNA was kept constant throughout the experiment by addition of appropriate amounts of the expression vector without insert. (B) MCF A/Z cells stably transfected with the 5-LOX expression vector (MCF A/Z-LOX) were transfected with the HIV-κB-CAT reporter plasmid alone or together with a FLAP expression vector, and CAT activities were measured in unstimulated cells or in cells stimulated with IL-1β as described for panel A. Asterisks indicate that values were statistically different from the reference values (Δ). (C) Formation of ROIs was measured by using DFCH in MCF7 A/Z cells transiently transfected with the FLAP expression vector. Column 1, background (no DFCH probe); column 2, unstimulated cells; column 3, stimulation with IL-1β (50 U/ml); column 4, stimulation with H₂O₂ (250 μM). (D) ROI production in MCF7 A/Z-LOX cells either unmodified or transiently transfected with the FLAP expression vector. Column 1, background (no DFCH probe); column 2, unstimulated cells; column 3, stimulation with IL-1β (50 U/ml); column 4, FLAP-transfected unstimulated cells; column 5, FLAP-transfected cells stimulated with IL-1β (50 U/ml); column 6, as in column 5 plus NAC (10 mM); column 7, as in column 5 plus NAC (20 mM); column 8, as in column 5 plus PDTC (60 μM); column 9, as in column 5 plus PDTC (100 μM); column 10, as in column 5 plus MK886 (0.5 μM); column 11, as in column 5 plus MK886 (1 μM); column 13, stimulation with IL-1β (50 U/ml); column 14, as in column 13 plus ETYA (35 μM); column 15, as in column 13 plus ETYA (65 μM); columns 12 and 16, stimulation with H₂O₂ (250 μM). Asterisks indicate that values were statistically different from the reference values (Δ).