Dickkopf-1 inhibits WNT3A-induced osteoprotegerin production in C2C12 cells. (A) Supernatants of breast cancer cells were harvested after 48 hours. C2C12 cells were cultured in control medium (containing 50% control L-cell medium) or differentiated in WNT3A-containing media (50%) and concurrently treated with breast cancer supernatants (25%) or unconditioned control media (25%). C2C12 cells were treated for 48 hours and assessed for osteoprotegerin (OPG) expression and protein secretion. (B) C2C12 cells were differentiated with WNT3A media and exposed to increasing concentrations of recombinant Dickkopf-1 (DKK-1; 10, 50, 100 and 250 ng/ml). (C) DKK-1 was transiently knocked down in MDA-231 cells using two different single interfering RNAs (siRNAs). Knockdown was verified by quantifying DKK-1 levels secreted into the supernatant 48 hours after transfection. Supernatants of control siRNA and DKK-1 siRNA-treated cells were given to C2C12 cells during WNT3A-induced differentiation in the established ratios of 25% and 50%. OPG levels were determined after 48 hours of exposure. (D) C2C12 cells were exposed to supernatants of untreated or zoledronic acid-treated MDA-231 cells in the presence of WNT3A. To prevent potential direct effects of zoledronic acid on C2C12 cells, MDA-231 cells were preincubated with zoledronic acid for 24 hours, media were then replaced and zoledronic acid-free supernatants were harvested 48 hours later. Suppressed DKK-1 levels in the supernatant were confirmed by enzyme-linked immunosorbent assay (ELISA) prior to use. Results of ELISA and polymerase chain reaction data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.