Fig. 3

GDF15 increases the recruitment of bone macrophages (osteomacs) via CCL2. GDF15 increases the expression of CCL2 in osteoblasts. a Total RNA was isolated from MCOs treated with rhGDF15 for 48 h and was assessed for CCL2 by RT–qPCR. b mRNA expression of CCL2 in MCOs after treatment (48 h) with CM from PC3, PC3 cells overexpressing GDF15, C4-2B cells, and C4-2B (GDF15 KO) cells with and without rhGDF15 in vitro. β-Actin was used to normalize the gene expression. c Immunohistochemical analysis of CCL2/MCP-1 in tibial sections from C4-2B- and C4-2B (GDF15 KO)-injected mice (right panel) quantification. Scale bar = 200 µm. d CCL2 levels in serum obtained from C4-2B- and C4-2B (GDF15 KO)-injected mice (n = 6 mice/group). e Serum CCL2 levels in PC3-Luc(i)- and PC3-GDF15(i)-injected mice (n = 6 mice/group). f Immunohistochemical analysis of CD68 on F4/80⁺ macrophages in bone sections obtained from C4-2B- and C4-2B (GDF15 KO)-injected tibiae; (right panel) quantification (n = 3 mice/group). Scale bar = 200 µm. The data are presented as the mean ± SEM, and P ≤ 0.05 was considered to indicate statistical significance. *P < 0.05, ***P < 0.001 compared with the control group and ^#P < 0.05, ^###P < 0.001 compared with the parental (C4-2B)-injected group