Rac2 promotes M2 macrophage polarization with no substantial change in macrophage recruitment.

<p>(A) Identification of F4/80⁺ macrophages by immunofluorescence microscopy in the frozen sections of LLC and B16 tumors stained with antibodies against F4/80 and imaged by fluorescence microscopy. The average no. of macrophages per HPF for 3 different experiments were 42±8, (WT) 38±6 (Rac2-/-) for LLC tumors and 50±5, (WT) 45±10, (Rac2-/-) for B16 tumors. Macrophages were counted blindly by 3 individuals in 5-10 randomly chosen fields and data is representative of three independent experiments with 4 mice. (B) Figure represents FACS data showing the quantification of CD11b and F480⁺ macrophages infiltrated in LLC tumors implanted in WT and Rac2-/- mice. Experiment was repeated 4-5 times with 3-4 mice in each group and similar results were obtained. (C) Quantitative PCR analysis of mRNA for M1, M2 specific genes in the macrophages sorted from LLC tumors grown in WT and Rac2-/- mice (n = 3–4) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095893#s4" target="_blank">Methods</a>. Values are mean ± SEM. Statistical significance is assessed by two sample <i>t</i>-test where *denotes <i>P</i><0.05, ** denotes <i>P</i><0.01 and *** denotes <i>P</i><0.001. (D) Arginase activity was measured in macrophages sorted from LLC tumors injected in WT and Rac2-/- mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095893#s4" target="_blank">Methods</a>. (E) Nitrite production in macrophages sorted from LLC tumors injected in WT and Rac2-/- and stimulated with 10 ng/ml LPS for 24 h. Supernatants were collected, and nitrite concentration was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095893#s4" target="_blank">Methods</a>. Results are mean ± SEM (n = 3–4 mice) for 3 independent experiments performed in triplicate (P<0.05 for arginase activity and <i>P</i><0.001 for nitrite assay; student's t test).