Fig. 6
GDF15 increases the osteoclastogenic potential of osteoblastic cells via an increased RANKL signal. a MCOs were treated with rhGDF15 for 48 h, and the mRNA expression levels of RANKL and OPG and the ratio of RANKL/OPG expression, representing the degree of osteoclastogenesis, were assessed. b RT–qPCR analyses showing the mRNA expression and ratio of RANKL and OPG in MCOs after treatment with CM from C4-2B cell lines (parental and GDF15 KO). c Immunohistochemistry of ALP and osteocalcin 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. d Immunohistochemistry of GFRAL and RET on the decalcified tibiae of PC3-injected mice. Scale bar = 200 µm. e RNA expression of GFRAL, RET, and Runx2 in MCOs cultured in normal (OB-1) and osteoblast differentiation medium (OB-2) using β-actin as a control. f MCOs were transfected with siRNA targeting GFRAL and control siRNA; after 48 h of transfection, the cell lysates were analyzed for GFRAL, pERK, pAKT, and the respective total forms. β-Actin was used as a loading control. g mRNA expression analyses of ALP, CCL2, and RANKL in MCOs after 48 h of GFRAL siRNA transfection. The data represent the mean ± SEM. P ≤ 0.05 was considered to indicate statistical significance
