NF-κB activation in MM cell lines induced by coculture with BMSCs. (A) Indicated MM cell lines were cultured with BMSCs for 12 hours and NF-κB activity was assessed by EMSA. The intensity of the bands was digitalized by ImageJ software and indicates fold increase compared with nontreated (control) cells after BMSC coculture. (B) To determine the role of Rel family proteins mediating NF-κB activity induced by coculture of MM.1S and OPM1 cells with BMSCs, supershift assays were carried out using anti-p65(65), p50(50), p52(52), RelB (B), and c-Rel (cR) Abs. (C) MM cells were cultured alone (C) as well as with BMSCs (A, adhesion) or with BMSC culture supernatant (S) for 12 hours. Nuclear extracts were subjected to EMSA to assess NF-κB activity. Exposure time of autoradiography varied for each cell line.