Knockdown DKK1 by shRNA restored Wnt3a-induced OPG in osteoblasts. C2C12 cells were transiently infected with supernatant containing control siRNA (shCont) or shRNA specific for DKK1 for indicated times. Total RNA was then isolated and subjected to RT-PCR for detecting DKK1 mRNA (A). cDNA from 24 hours was subject to qPCR to confirm DKK1 mRNA expression (B). Supernatants of the cells were harvested and subjected to ELISA for measuring DKK1 protein (C). The infected cells were treated with rWnt3a for 48 hours and RNA and supernatants were harvested and subjected to qPCR and ELISA analysis for OPG mRNA (D) and protein (E). Data represent the means plus or minus SD (n = 3) of representative experiments (**P < .01, ***P < .001, vs control).