Effects of TNFα on mRNA levels. (A) mRNA samples of freshly isolated neutrophils and neutrophils incubated in the presence of TNFα (10 ng/mL) for 3 hours were determined using Atlas nylon arrays. Selected spots shown are those of caspase-3, MCL-1, and BFL-1. Typical result of 1 of 3 separate experiments using neutrophils from different donors. (B) HeLa cells were transfected with luciferase vectors driven by either the BFL-1 or MCL-1 promoters. Twenty-four hours after transfection, the cells were incubated in the presence or absence of GM-CSF (50 U/mL), TNFα (10 ng/mL), or PMA (10 μg/mL) with (▩) or without (■) the NF-κB inhibitor parthenolide (10 μM). Values shown are means (± SD, n = 3). (C) Neutrophils were incubated in the presence of TNFα (10 ng/mL), and RNA samples were isolated at the indicated time points. Levels of transcripts for BAX (■), BFL-1 (●), and MCL-1 (○) mRNA were measured using quantitative real-time PCR (normalized to GAPDH levels). Values shown are means (± SD, n = 3).