Stable PCNA protein levels are maintained in neutrophils exposed to G-CSF in vitro without transcriptional regulation. (A) Neutrophils were cultured with and without 1,000 U/ml G-CSF at 37°C. After 0, 3, 6, or 15 h of incubation, cells were lysed, and Western blot analysis was performed with the PC10 mAb, and anti–β-actin served as the loading control. (B) Neutrophils were incubated for 15 h at 37°C in the absence (0) or in the presence of 200 or 1,000 U/ml G-CSF and analyzed as described in A. The percentages of viable neutrophils, i.e., annexin-V⁻ 7-AAD⁻ cells under the same conditions, are shown in the histogram below the blot. (C) Total RNA was extracted from neutrophils cultured with and without 1,000 U/ml G-CSF for the times indicated, and PCNA, BLyS, and β2m mRNA expression was measured by real-time RT-PCR and primary transcript real-time RT-PCR. Their expression is given as mean normalized expression (MNE) after normalization to β2m in triplicate reactions for each sample. Error bars represent SEM. The data presented in A–C are from one representative experiment of three.