Effect of blocking NF-κB activation on TNF-α–induced TM repression. HUVECs were pretreated with parthenolide at the indicated doses or transduced with either AdNull or AdIκBsr (MOI [multiplicity of infection] = 100 pfu/cell) prior to stimulation with or without 100 ng/mL human TNF-α. (A) NF-κB activity measured in nuclear extracts by ELISA 1 hour after TNF-α stimulation. (B) TM gene expression, normalized to CD31, was determined by real-time PCR 16 hours after TNF-α stimulation. (C) TM protein expression was determined by Western blot analysis 16 hours after TNF-α stimulation. Representative blots are shown at bottom, and densitometric analysis with normalization to actin is shown at top. Par indicates parthenolide. Values in all figures are the mean ± SEM of 3 experiments (n = 5-6 for control groups). ▪ indicates vehicle only; ▦ indicates stimulation with TNF-α. ^*P ≤ .02 for 8 μM parthenolide compared with 2 μM parthenolide or control groups stimulated with TNF-α. #P ≤ .03 for AdIκBsr compared with AdNull or control groups stimulated with TNF-α.