Effect of myeloma cells on Runx2/Cbfa1 expression and activity by PreOBs. (A) The expression of Runx2/Cbfa1 mRNA was evaluated by RT-PCR in PreOBs after 24-hour coculture with OPM2 in the presence or absence of a transwell insert. Nuclear extracts (80 μg) were obtained from PreOBs, PreOBs cocultured for 48 hours with OPM2 placed in a transwell insert, or from PreOBs pooled with OPM2. The expression of Runx2/Cbfa1 protein was analyzed by Western blot as described in “Patients, materials, and methods.” (B) Runx2/Cbfa1 was identified as a band of a molecular weight of about 65 kDa. (C) Human osteoblast cell line HOBIT was used as positive control. Runx2/Cbfa1 DNA-binding activity was evaluated by EMSA. Nuclear extracts (20 μg) of the cells were incubated with 32P-labeled OSE2 probe. Protein-DNA complexes were resolved in 5% nondenaturating polyacrylamide gel, which was then dried by heating in vacuum and analyzed by autoradiography. (Ci) To validate the EMSA protocol we used HOBIT (positive control), HOBIT plus 100-fold wild-type unlabeled probe (competitor), HOBIT plus 100-fold mutant unlabeled probe (nonspecific competitor), HOBIT plus antibody specific for Runx2/Cbfa1 (supershift). (Cii) A coculture system between PreOBs and the HMCLs OPM2 and XG-1, healthy BMMNCs, EBV cell line B95.8, or freshly purified MM cells of a representative patient (MM1) has been performed for 24 hours, and Runx2/Cbfa1 DNA-binding activity was evaluated by EMSA. (Ciii) In some experiments, OPM2 and fresh MM cells (MM1) were cocultured in the presence or absence of a transwell insert. (Civ) Finally, PreOBs and fresh MM cells (MM1) were cocultured in a cell-to-cell contact system with or without blocking anti-VLA-4 mAb or anti-IgG control mAb. Figures are representative of 3 independent experiments.