VCAM-1 expression, IL-6 production, and downstream signaling of NF-κB and p38 MAPK by stromal cells from MM patients and healthy subjects. (A) Stromal cells from healthy and MM patients were cultured with 10 ng/mL TNF-α for 4 days, the cell lysates were collected, and the levels of VCAM-1 were determined by Western blot analysis using anti–VCAM-1 antibody. Densitometry readings were all compared with healthy patient 1 untreated stromal cells. (B) Activation of downstream signaling pathways in normal or MM stromal cells induced by TNF-α. Stromal cells (5 × 10⁴ cells/well) from healthy subjects and MM patients were cultured with 10% FCS in IMDM for 14 days. Cells were then cultured with 2% FCS in IMDM for 24 hours as a means of starvation. After starvation, cells were exposed to TNF-α (10 ng/mL) for the indicated times. Cells were lysed, fractionated by SDS-PAGE, and analyzed by immunoblot using antibodies recognizing phosphorylated and total signaling molecules of IκBα and p38 MAPK. β-Actin served as the loading control. (C) NEMO inhibitor but not p38 inhibitor decreases TNF-α–induced VCAM-1 expression in MM stromal cells. Stromal cells from MM patients were cultured for 4 days with TNF-α in the presence or absence of 10 μM NEMO inhibitor or 10 μM of a p38 MAPK inhibitor (SB203580). The cell lysates were collected and analyzed by Western blot analysis using anti–mouse VCAM-1. Densitometry reading for healthy subjects and MM patients were compared independently to their relevant MM or normal controls. (D) IL-6 production from MM stromal cells was blocked by the p38 MAPK inhibitor. Stromal cells from MM patients were cultured for 4 days with TNF-α and/or NEMO and p38 MAPK inhibitors. The culture media were collected, and IL-6 was measured with an IL-6 ELISA kit. *P < .01 compared from TNF-α alone. NS indicates not significantly different from vehicle treated cells. Similar results were seen in 6 independent experiments using MM and normal derived primary stromal cell samples.