Effects of EGCG on NO-induced NF-κB activation in HEI-COΙ cells.

<p>(A) After transfection, cells were treated with 500 μM SNAP for 4 h. After isolation of the nuclear fraction, protein extracts were assayed for NF-κB activity by western blot analysis. (B) Protein extracts were assayed for caspase-3 by western blot analysis. (C) Relative levels of NF-κB and caspase-3 are shown. (D) Cells were pretreated with 50 μM EGCG, followed by treatment with 500 μM SNAP for 4 h. After isolation of cytosolic and nuclear fractions, protein extracts were assayed for IκB-α and NF-κB by western blot analysis. (E) Cells were transfected with an NF-κB-dependent reporter gene for 48 h, and transfected cells were treated with SNAP for 4 h. EGCG (50 μM) was added 2 h prior to SNAP treatment. Cells were harvested, and luciferase activity was measured as described in the Materials and Methods. (F) Cells were fixed and stained with NF-κB (green) and DAPI (blue). Cells were then analyzed using an Olympus microscope (magnification, 100×). All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).