Figure 2. The activation of NF-κB is essential for oleate-induced PTX3 expression.

(A and B) TU183 cells were treated with 400 μM oleate for the indicated period of time. The protein levels were determined by Western blotting with antibodies against AKT, IκBα, β-actin and phosphorylated AKT and IκBα. The expression of IkBα was calculated by image-based computational quantification (A). The cytoplasmic fractions and nuclear extracts of the cells were prepared in the same volume of buffer, and an aliquot of each fraction was used for Western blotting analysis using antibodies against NF-κB, HDAC-1 and α-Tubulin (B). (C) Cells were pretreated with 20 μM LY294002 (Sigma-Aldrich, St Louis, MO, USA) for 1 h, then with 400 μM oleate for 1 h (i) or 6 h (ii). The protein levels in cell lysates were determined by Western blotting with antibodies against AKT, PTX3, phosphorylated AKT and β-actin. (D and E) Cells were transfected with the DN-IκB expression vector by lipofection or pretreated with 10 μM parthenolide (Sigma-Aldrich, St Louis, MO, USA) or 5 μM U0126 (Sigma-Aldrich, St Louis, MO, USA) for 1 h, then with 400 μM oleate for 1 h (i) or 6 h (ii) (D). The protein levels in cell lysates were determined by Western blotting with antibodies against ERK, PTX3, IκBα, β-actin and phosphorylated IκBα and ERK. (F and G) Cells were treated with 400 μM oleate for 1 h and then treated with 4 μM actinomycin D (Sigma-Aldrich, St Louis, MO, USA) for the indicated period of time (F). On the other hand, cells were transfected with the DN-IκB expression vector by lipofection or treated with 10 μM parthenolide and 400 μM oleate for 1 h, and then treated with 4 μM actinomycin D for 3 h (G). The expression of PTX3 mRNA was analyzed by real-time quantitative PCR. Relative levels of PTX3 were normalized by GAPDH and the degradation rate was calculated by comparing to 0 h (considered as 100%). The values represent the mean ± s.e.m. of three determinations.