Cell death in response to TNF and NF-κB inhibition are restored in myeloid leukemia cells by HO-1 siRNA and is caspase-dependent. (A) Where indicated, THP-1 and HL60 were untransfected, transfected with control siRNA, or with 3 different HO-1 siRNAs for 24 hours and then treated with 10 μM BAY 11-7082 and 10 ng/mL TNF for 8 hours. Protein expression was measured for HO-1 by Western blot analysis. (B) THP-1 cells and HL60 cells were transfected with control siRNA or HO-1 siRNA-2 before treatment with 10 μM of BAY 11-7082 in conjunction with various concentrations of TNF for 24 hours. Cell number was then assessed by MTS assay as described in “Proliferation/death and apoptotic assays.” Values represent mean plus or minus SEM from 3 independent experiments. (C) Apoptotic cells detected by epifluorescence after staining with Hoechst 33342. (D) THP-1 cells and HL60 cells were transfected with control siRNA or HO-1 siRNA-2 before treatment with 50 μM of the caspase inhibitor of zVAD-fmk. Cells were then treated with BAY 11-7082 in conjunction with TNF for 24 hours. Cell number was then assessed by MTS assay as described in “Proliferation/death and apoptotic assays.” Values represent mean plus or minus SEM from 3 independent experiments (*statistical significance, P < .01, between the different treatment groups).