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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 5. — CCL3 is required for MAMs to accumulate in tumor-challenged lung. (A) Relative numbers of lung CD11b⁺ macrophages (left) or circulating CD11b⁺CD115⁺ monocytes (right) in C57BL/6 (WT; n = 14), Ccl3^−/− (n = 4), and Ccr2^−/− (n = 4) mice (normal; N) were compared with those i.v. injected with E0771-LG cells 24 h before measurement (tumor; T). IM, inflammatory monocytes (Ly6C⁺); RM, resident monocytes (Ly6C^–). Data are measured as percentage of macrophages or monocytes in CD45⁺ cells (at least four independent experiments). Data are means ± SEM. *, P < 0.05 versus WT-N; ^†, P < 0.05 versus WT-T. Gating strategy is shown in Fig. S1. (B) Relative numbers of leukocytes in lungs of C57BL/6 (WT; n = 14), Ccl3^−/− (n = 4), and Ccr2^−/− (n = 4) mice are shown. Mice were injected with PBS (Normal) or E0771-LG cells (Tumor) 24 h before measurement. Rmac, resident macrophages; Neu, neutrophils. Data are measured as percentage of macrophages or monocytes in CD45⁺ cells (at least four independent experiments) and shown as means ± SEM. *, P < 0.05 versus WT-Normal. (C) Relative numbers of circulating neutrophils (Neu) and inflammatory monocytes (IM) in WT mice 24 h after 2 d of treatment with anti-Ly6G antibody (n = 5, two independent experiments). Data are measured as percentage of macrophages or monocytes in CD45⁺ cells. Data are means ± SEM. *, P < 0.01. (D) Number of lung foci was assessed in neutrophil depleted WT mice at day 11 after E0771-LG breast cancer cell injection. Mice were treated with IgG or anti-Ly6G antibodies 24 or 5 h before, and 16 h after tumor cell injection (n = 4, four independent experiments). Data are means ± SEM. No statistical significance was observed. (E) Relative number of circulating monocytes was assessed in rtTA:tetO-Cre:Csf1r^F/F mice treated with or without doxycycline (Dox) in drinking water for 7 d (n = 4, 2 independent experiments). Data are means ± SEM. *, P < 0.01. (F) Number of metastasis foci was assessed in monocyte-depleted rtTA:tetO-Cre:Csf1r^F/F mice at day 11 post-tumor injection. The rtTA:tetO-Cre:Csf1r^F/F mice injected with E0771-LG cells were given water with or without doxycycline (Dox) in drinking water from 7 d before tumor injection to the endpoint (n = 7, 2 independent experiments). Data are means ± SEM. *, P < 0.01.