Osteoclasts and MSCs down-regulated JUN and EGR-1 in myeloma plasma cells. (A-C) Primary myeloma plasma cells from 8 patients were cultured in the presence (coculture) or absence (pre-coculture) of osteoclasts for 2 days. (A) JUN and EGR-1 gene expression levels in these cells were evaluated by real-time PCR, expressed as relative values by the comparative Ct method. (B) Protein levels of JUN and EGR-1 were determined by Western blot analysis in 3 patient samples, shown in panel A, that had sufficient quantities of protein extracts available. Protein levels were compared in cultured cells in the presence (co) or absence (pre-co) of osteoclasts. β-Actin was used as a loading control. (C) JUN binding activity was evaluated in nuclear extracts of the same 3 samples of myeloma plasma cells shown in panel B by the use of an ELISA-based assay (expressed as relative value compared with each control); each sample is expressed as the mean ± SEM of 2 duplicates. JUN (D) and EGR-1 (E) gene expression from GEP data of myeloma cells cultured with (t = 18 hours) or without (t = 0 hours) MSCs.