Figure 3. In vitro effects of PACA on macrophage polarization. (A) Effects of PACA on iNOS and COX-2 expression in LPS-stimulated macrophages. Following 2 h pretreatment with PACA, RAW264.7 macrophages were co-stimulated with PACA and LPS for another 24 h. The cellular proteins were analyzed by Western blotting for the expression of iNOS and COX-2 (n = 3). **, p<0.01, ***, p<0.001 (PACA+LPS vs LPS alone). (B) Flow cytometric analysis of macrophage biomarker CD80 and CD163. Following 2 h pretreatment with PACA, RAW264.7 macrophages were co-stimulated with PACA, LPS and/or IFN-γ for another 24 h. Macrophages were stained with antibodies against CD80 and CD163 and analyzed by flow cytometry. (C) qRT-PCR analysis for the mRNA levels of macrophage biomarkers. After the treatment as described in Panel B, the total RNAs was isolated and analyzed by qRT-PCR technique. Pro-inflammatory macrophage biomarkers included iNOS, CXCL10, IL-6, CCL2, IL-1β and TNF-α whereas pro-resolving macrophage biomarkers included Arg1, IL-10, FZZ1 and Ym-1 (n = 3). **, p<0.01, ***, p<0.001 (PACA+LPS vs LPS).