Figure 3

MM BMSC-induced NF-κB activity is mediated by a secreted peptide factor.(A) EMSA of NF-κB activity in RPMI8226 cells cultured alone or with BMSCs from the indicated MM patients, either directly, or physically separated by a Transwell insert ("Transwell"). 10 ng/mL TNF-α was placed either directly in RPMI8226 culture or below the Transwell insert as a positive control for membrane permeability. The black line indicates a different exposure time for the same gel for TNF-α-treated samples in order to better visualize the very different levels of binding activity. (B) EMSA of NF-κB and Oct-1 DNA-binding activity in RPMI8226 cells treated with 1× conditioned media (CM) or the same CM concentrated 2-fold (2×) or 5-fold (5×) for 24 hours. (C) EMSA of NF-κB activity in primary MM cells treated with CM derived from BMSCs of the indicated MM patients. (D) EMSA of NF-κB and Oct-1 DNA-binding activity in RPMI8226 cells treated with CM that had been previously treated with proteinase K ("P"), or heat ("H") where indicated. RPMI8226 cells were treated with CM from Pt. 134, and 100 nM bortezomib was added for the last 4 hours of culture where indicated ("B"). NF-κB dimers are labeled as before.