TNF in conjunction with NF-κB inhibition induces HO-1 gene expression in myeloid leukemia cells but not primary cells. (A) Primary monocytes, THP-1 cells, and HL60 cells were treated with either BAY 11-7082 (10 μM or transfected before treatment with TNF (10 ng/mL) for the indicated time. RNA was extracted and after reverse transcription, HO-1 mRNA expression was measured by real-time PCR. (B) HO-1 protein expression was measured by Western blot analysis. Cells were treated with 10 μM BAY 11-7082 30 minutes before 8 hours of treatment with 10 ng/mL TNF as indicated. (C) Primary monocytes, THP-1 cells, and HL60 cells were pretreated with 10 μM BAY 11-7082 30 minutes before treatment with TNF (10 ng/mL) for 4 hours. After reverse transcription, IL-1β mRNA expression was measured by real-time PCR. (D). Primary monocytes, THP-1 cells, and HL60 cells were transfected with p65 siRNA 24 hours before stimulation with TNF (10 ng/mL) for 4 hours. HO-1 mRNA expression was measured by real-time PCR. (E) THP-1 cells were pretreated for 30 minutes with either curcumin (1 μM) or MG132 (5 μM) or transfected 24 hours previously with 0.5 mg of a Iκ-Bα dominant negative mutant (IκBα-DN) construct before stimulation with TNF (10 ng/mL) for 4 hours. HO-1 mRNA expression was measured by real-time PCR. Values indicate mean plus or minus SEM from 3 independent experiments (*significance, P < .01, between the different treatment groups).