Macrophages deficient in Dock180 fail to form MNGCs. (A) (i) Brag2 (1,100 bp) and Dock180 (745 bp) expression in thioglycollate-induced IP-harvested macrophages before and after treatment with IL-4 as indicated by RT-PCR analysis. β-actin (300 bp). (ii) Semi-quantitative RT-PCR showing expression of Dock180 in CD11b⁺Gr1⁺ monocytes obtained from GFP⁺ peripheral blood of PVR-Cnt-BM and PVR-Dock180Si-BM mice 4 wk after transplantation. (iii) Semi-quantitative RT-PCR showing expression of Dock180 in CD11b⁺ IP-macrophages of PVR-Cnt-BM and PVR-Dock180Si-BM mice 6 wk after transplantation under fusion conditions. (B) Phase-contrast and immunofluorescence images of IP-harvested macrophages from: (i) PVR-Cnt-BM macrophages serving as negative fusion control cultured without IL-4; (ii) GFP⁻ cells from PVR-Dock180Si-BM representing the endogenous macrophage population cultured with IL-4; (iii) GFP⁺ cells from PVR-Cnt-BM representing the transplanted control population cultured with IL-4; and (iv) GFP⁺ cells from PVR-Dock180Si-BM representing the transplanted Dock180-deficient population cultured with IL-4. MNGC formation can be distinguished by the cluster of nuclei with a large surrounding area of cytoplasm, the extent of which is indicated by dashed lines. (C) Fusion index analysis in macrophages from each of the four populations shown in B. Two multinucleated categories are analyzed and a minimum of 1,000 nuclei were counted for each fusion assay. Error bars indicate the mean ± SE of four independent fusion assays. P values were determined by t test between the two positive fusion controls and the PVR-Dock180Si-BM macrophages (*, P < 0.03; **, P < 0.0005). (D) Comparison of fusion indexes, of three or more nuclei, in myoblasts and macrophages deficient in Dock180. Error bars indicate the mean ± SE of four independent fusion assays (*, P < 0.00001; **, P < 0.0005).