FIGURE 3.

Adipocyte conditioned medium inhibits osteoclastogenesis. (A) Experimental design of the in vitro assay used to test anti-osteoclastogenic activity from mature adipocyte secreted factors. (B) One day after plating, wild type BM cells were cultured for 6 more days in the presence of M-CSF and RANKL (osteoclast differentiation media) in addition to conditioned medium prepared from differentiated (ACM, adipocyte-conditioned medium) or undifferentiated (CTL: control-conditioned medium) 3T3-L1 preadipocytes. Different doses of a specific blocking antibody against adiponectin (1.5–12.0 μg/ml) were added to the conditioned medium (ACM50) during osteoclast differentiation (ACM 100 means pure ACM; ACM50 means 1:1 diluted ACM with osteoclast differentiation medium). Data are presented as mean ± SEM of biological triplicates. Statistical significance was determined by two-tailed unpaired t-test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (C) Calvaria resorption pit assay. Representative sections of skulls from 14 day-old wild-type pups cultured for 2 weeks in complete αMEM in the presence of conditioned medium from either undifferentiated 3T3-L1 preadipocytes (CTL, n = 3) or mature 3T3-L1 adipocytes (ACM, n = 3), after TRAP staining. Top panel: the white arrow indicates one example of resorption pit seen in many places on the skull with CTL medium, while absent with the ACM culture medium. (D) Western-blot analysis of conditioned medium from undifferentiated (CTL) and differentiated 3T3-L1 adipocyte (A-CM) against adiponectin. Recombinant mouse adiponectin (200 ng rmAdiponectin) was added to demonstrate specificity of the signal.