Osteoclasts down-regulate DKK1 and JNK pathway components in myeloma plasma cells. Primary myeloma plasma cells were cultured in the presence (⊡) or absence (▪) of osteoclasts for 2 days. (A) Microarray analysis was used to determine expression of DKK1, FOS, FOSB, c-JUN, and JUND in 8 samples with high baseline DKK1 expression (expressed as Affymetrix signal, 1000-unit increments). (B) DKK1 expression was evaluated by real-time PCR in another 9 samples cultured with (⊡) or without osteoclasts (▪), expressed as relative values using ΔΔCt method. (C) c-Jun–binding activity was evaluated in nuclear extracts of the same 9 samples of myeloma plasma cells shown in panel B, using an ELISA-based assay (expressed as relative value compared to each control). In panels B and C, each sample is expressed as the mean ±SEM of 2 duplicates. (D) The level of activation of the JNK pathway was determined by Western blot analysis for the phosphorylated isoforms of p46 JNK1 (p-p46) and p54 JNK2 (p-p54) in cytosolic extracts of 4 samples shown in panel C that had sufficient quantities of protein extracts available. Number 1 represents control samples; number 2 represents samples after osteoclast cocultures. Note that the modulation in JNK pathway activation is not due to variation in total levels of the JNK isoforms.