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. 2015 Jun 29;212(7):1043–1059. doi: 10.1084/jem.20141836

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© 2015 Kitamura et al.

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Figure 2. — CCR2 signaling regulates CCL3 expression in MAMs. (A) Levels of Ccl3 mRNA (left) and CCL3 protein (right) in BMDMs isolated from WT or Ccr2^−/− mice were assessed by RT-PCR (n = 6 per genotype, 6 independent experiments) and ELISA (n = 3 per genotype, two independent experiments). Data are means ± SEM. *, P < 0.01 versus WT. (B) Ccl3 mRNA (left) and CCL3 protein (right) were assessed by RT-PCR (n = 3, 2 independent experiments) and ELISA (n = 3 per genotype, 2 independent experiments) in macrophages cultured with PBS or recombinant CCL2 (100 ng/ml). The BMDMs were isolated from WT or Ccr2^−/− mice. Data are means ± SEM. *, P < 0.01. (C) Expression of Ccl3 mRNA was assessed by RT-PCR (n = 4, four independent experiments) in monocytes and macrophages obtained from E0771-LG tumor-injected mice treated with neutralizing anti-CCL2 or control IgG antibodies. IM, inflammatory monocytes (CD115⁺CD11b⁺Ly6C⁺); RM, resident monocytes (CD115⁺CD11b⁺Ly6C^–); MAMs (F4/80⁺CD11b⁺CD11c^–Ly6C^–); Rmac, resident macrophages (F4/80⁺CD11b^–CD11c⁺Ly6C^–). Data are means ± SEM. *, P < 0.01 versus IgG-IM; ^†, P < 0.05 versus IgG-MAM. Gating strategies are shown Fig. S1. (D) Expression of Ccl3 mRNA was assessed by RT-PCR in Rmac, MAM (n = 7, 7 independent experiments) or neutrophil (Neu), CD4⁺ T cell, CD8⁺ T cell, B cell, and NK cell (n = 3, 3 independent experiments). The leukocytes were isolated from tumor-bearing mouse lung. Data are means ± SEM. *, P < 0.01 versus Rmac. (E) Relative CCL3 mRNA expression was assessed by RT-PCR (n = 4, 4 independent experiments) in human monocytes (Mo) and those differentiated into macrophages by CSF-1 (Mac). Data are means ± SEM. *, P < 0.05. (F) Relative CCL3 mRNA level was assessed by RT-PCR (n = 4, 4 independent experiments) in human macrophages cultured with recombinant human CCL2 (100 ng/ml). Data are means ± SEM. *, P < 0.05. (G) Expression of CCL3 mRNA was assessed by RT-PCR in human MDMs (hMDM) cultured with conditioned medium (C.M.) from human cancer cell lines as indicated. As a control (–), the cells were cultured in nonconditioned medium (αMEM including 10% FBS and 10³ U/ml CSF-1). n = 5, 5 independent experiments. Data are means ± SEM. *, P < 0.01. (H) Expression of CCL3 mRNA was assessed by RT-PCR in human macrophages cultured in MDA231 conditioned medium with neutralizing anti-CCL2 or control IgG antibodies. n = 3. Data are means ± SEM. *, P < 0.05.