Effects of knockdown of p62 in marrow stromal cells. (A) p62 siRNA or control RNA transduced normal or MM stromal cells were cultured for 3 days, serum-starved for 24 hours and then exposed to serum for 10 minutes, and the cell lysates from the stromal cells were collected. Western blot analysis was performed using anti-p62 (BD Biosciences), anti–phosho-PKCζ, anti-PKCζ, and anti-VCAM-1, and the relative levels compared with the appropriate control were determined by densitometry. (B) IL-6 concentrations in culture media were measured after 3 days of culture by IL-6 ELISA assay kit. (C) Activation of downstream signaling pathways in p62 siRNA transduced MM stromal cells induced by TNF-α. p62 siRNA or control RNA transduced MM stromal cells (5 × 10⁴ cells/well) were cultured with 10% FCS in IMDM for 2 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, NF-κB, and p38 MAPK. β-Actin served as the loading control. All antibodies were purchased from Cell Signaling. (D) p62 siRNA (0-10 mg) or control siRNA transduced normal or myeloma-derived stromal cells were cocultured with the ANBL-6 MM cell line for 3 days. MM cell growth was determined by counting CD138⁺ MM cells after removing the MM cells by vigorously pipeting. (E) Stromal cells were cocultured with ANBL-6 as described in “Methods.” The conditioned media were collected after 3 days, and the IL-6 concentrations were measured by ELISA kit. Similar results were seen in 3 independent experiments.