GM-CSF significantly reduces Bad transcription in human neutrophils.

Human neutrophils were incubated in the presence or absence of GM-CSF (10 ng/mL) or TNF-α (200 U/mL) with or without LY294002 (10 μM). Total RNA was isolated using TRIzol, and RT-PCR was carried out as described in the “Materials and methods” section. (A) Neutrophils incubated for 4 hours with GM-CSF showed a PI3-kinase–dependent decrease in the amount of Bad mRNA. Incubation with TNF-α led to a PI3-kinase–dependent increase in Bad mRNA at 4 hours (B) and a decrease in Bad mRNA at 20 hours (C). (D) Equal amplification of β-actin transcripts confirmed identical total RNA content of the samples at 4 hours (20-hour data not shown for clarity). Single representative immunoblots and corresponding densitometric analysis are shown. Corresponding immunoblots demonstrate the amount of total Bad present in neutrophil whole-cell lysates at these times. The data in panel A represent the mean ± SEM of 6 independent experiments.