Coculturing osteoblast cells with DKK1- expressing MM cells inhibited Wnt3a-induced OPG. A MM cell line OPM-2 was transfected with pEF6 vector (designated OPM-2/EV) or the vector containing Dkk1 cDNA (OPM-2/DKK1). DKK1 protein from OPM-2/EV or OPM-2/DKK1 cell lysates of selected clones was determined by Western blot analysis using anti-V5 antibody as described in “Immunoblotting analysis and GST-E cadherin binding assay” (A). The concentration of DKK1 protein in culture supernatants in OPM-2/EV and OPM-2/DKK1 cells was measured by ELISA analysis (B). C2C12 cells were cocultured with OPM-2/EV or OPM-2/DKK1 cells in the presence of rWnt3a or control for the indicted times. OPG synthesis in these cells, as measured by qPCR, is presented (C). Supernatants of the C2C12 were harvested and subjected to ELISA analysis to measure OPG protein concentration (D). The results are shown as means plus or minus SD (n = 4). Results are representative of 3 independent experiments (**P < .01, ***P < .001, vs control). C2C12 cells were cultured with primary CD138-positive plasma cells from 2 MM patients (P#1 and P#2) for 48 hours in the presence or absence of rWnt3a for 48 hours. The OPG mRNA in C2C12 cells was determined by qPCR (E). OPG protein levels were measured by ELISA analysis (F).