DKK1 and sera from MM patients inhibits Wnt3a-induced suppression of RANKL in osteoblast. C2C12 (A), Saos-2 (B), and MG63 (C) cells were treated with rWnt3a or Wnt3a-CM (as indicated) or Cont-CM for 48 hours after prior treatment with 100 ng/mL of DKK1 protein for 2 hours. RANKL mRNA was analyzed by qPCR. C2C12 cells transfected with empty vector (pEF/EV) or the vector carrying DKK1 cDNA (pEF/DKK1) were cultured in presence or absence of rWnt3a protein (100 ng/mL). The RNA and supernatant were harvested and subjected to qPCR analysis to determine RANKL mRNA (D) or ELISA to measure RANKL protein (E). C2C12 cells were treated with rWnt3a protein for 48 hours after prior incubation with sera (50% diluted with fresh serum free DMEM medium) from MM patients (n = 8) containing low (< 10 ng/mL) or high concentration of DKK1 (> 100 ng/mL) or normal sera (50% diluted with serum free DMEM medium) for 2 hours (F). Total RNA was isolated and subjected to cDNA synthesis. RANKL mRNA was amplified by qPCR analysis. The results are shown as means plus or minus SD (n = 3; *P < .01, **P < .001, vs control).