XN down-regulates the TNF-induced NF-κB activation induced by different agents and in different cell lines. (A) Time-dependent effect of XN on TNF-induced NF-κB activation. KBM-5 cells were preincubated with 50 μM XN for the indicated times and then treated with 0.1 nM TNF for 30 minutes. Nuclear extracts were prepared and assayed for NF-κB activation using EMSA. The fold activation of NF-κB and cell viability (CV) are shown at the bottom. (B) Dose-dependent effect of XN on TNF-induced NF-κB activation. KBM-5 cells were incubated with XN at the indicated concentrations for 4 hours and treated with 0.1 nM TNF for 30 minutes. Nuclear extracts were assayed for NF-κB activation using EMSA. The fold activation of NF-κB and cell viability (CV) are shown at the bottom. (C) XN inhibits NF-κB activation induced by CSC, H₂O₂, IL-1β, PMA, epidermal growth factor (EGF), LPS, okadaic acid (OA), and TNF. KBM-5 cells were preincubated with 50 μM XN for 4 hours and then treated with 0.1 nM TNF for 30 minutes, 500 nM okadaic acid for 4 hours, 250 μM/mL H₂O₂ for 2 hours, 25 ng/mL PMA for 2 hours, and 10 μg/mL LPS, 10 μg/mL CSC, and 100 nM IL-1β for 1 hour each. Nuclear extracts were analyzed for NF-κB activation using EMSA. The fold activation of NF-κB is shown at the bottom. (D) Effect of XN on constitutive NF-κB activation. Multiple myeloma U266 cells were incubated with XN at the indicated concentrations for 4 hours. Nuclear extracts were prepared and analyzed for NF-κB activation by EMSA. The fold activation of NF-κB is shown at the bottom. (E) Effect of XN on TNF induced NF-κB activation in other types of leukemia cells. K562, HL-60, U937, and Jurkat cells were incubated with XN at the indicated concentrations for 4 hours and treated with 0.1 nM TNF for 30 minutes. Nuclear extracts were assayed for NF-κB activation using EMSA.