The inhibitory effect of IPA-3 on NF-κB nuclear translocation.

(A) Effect of IPA-3 on subcellular localization of NF-κB was evaluated by immunofluorescence staining. After overnight serum starvation, H2M (left panel) and MIHA (right panel) cells were treated with either DMSO or IPA-3 (20 µM, 15 minutes) followed by an addition of TNF-α (20 ng/ml, 15 minutes). NF-κB was detected with a specific antibody (Green) and nucleus was stained with DAPI (Blue). (B) Western blotting analysis of P-PAK1 (T423) and total PAK1 were detected in the H2M cells stimulated by TNF-α (20 ng/ml) with or without IPA-3 pretreatment (20 µM, 15 minutes). TNF-α was included in the culture medium for 0, 0.5, 1, 2 or 4 hours. (C) Expression of quantitative real-time PCR was performed to analyze the mRNA level of MMP-9 (left panel) and COX-2 (right panel). Serum-starved H2M cells were treated with or without IPA-3 pretreatment (10 or 20 µM, 15 minutes) followed by TNF-α (10 or 20 ng/ml, 24 hours). Quantitative results of MMP-9 and COX-2 mRNA levels were normalized to β-actin. The values represented the mean ± SD of three independent experiments. *P<0.001 (ANOVA), ***P<0.05 (ANOVA) compared with the TNF-α control.