TNF in conjunction with NF-κB inhibition induces Nrf2 activation. THP-1 cells and HL60 cells were untreated or treated with 10 μM BAY 11-7082, or 10 ng/mL TNF, or BAY 11-7082 plus TNF, for the indicated times and nuclear extracts prepared. (A) Western blot analysis was carried out using anti-Nrf2 and anti–β-actin antibodies. (B) Samples treated where indicated with 10 μM BAY 11-7082 30 minutes before treatment with TNF (10 ng/mL) for a further 1 hour. EMSA reactions were performed using biotinylated double-stranded oligonucleotides corresponding to the human HO-1 ARE. (C) An unbiotinylated HO-1 ARE probe was included as a cold competitor (as indicated). Nuclear extracts from THP-1 cells untreated or treated with TNF in conjunction with NF-κB inhibition were left alone or preincubated with 1 μg anti-Nrf2 or anti-p65 (NF-κB) supershift antibodies before EMSA. (D) THP-1 cells and HL60 cells were loaded with 5 μM of DCF-DA before treatment with 10 μM BAY 11-7082, or 10 ng/mL TNF, or BAY 11-7082 plus TNF. Cells were then assessed using flow cytometry as described in “Proliferation/death and apoptotic assays.” Mean fluorescence intensity (MFI, arbitrary units) of the generation of ROS is measured. (E) THP-1 cells and HL60 cells were pretreated with 10 mM NAC before treatment with BAY 11-7082 plus TNF. Western blot analysis was carried out using anti-Nrf2, anti HO-1, and anti-β-actin antibodies. Results are representative of similar findings from at least 3 separate experiments.