Potentiation of neutrophil apoptosis by carboxyp21, a PCNA-competing peptide. The synthetic carboxyp21 and a control modified p21 peptide (whose charged amino acids, identified as crucial for binding to PCNA, were modified to prevent its binding to PCNA) were incubated with neutrophils to evaluate their effect on apoptosis. (A) Structure of PCNA (blue, red, and gray are used to distinguish the three monomers) bound to two carboxyp21 (yellow) and one modified p21 peptide (green). The structure represented was obtained at the end of the MD simulation. The secondary structure elements are highlighted by a ribbon representation, and two different views are presented: from above the ring (left) and from the side (right). (B) Neutrophils were incubated with 50 µM FITC-conjugated carboxyp21 peptide for 1 h and then analyzed by fluorescence microscopy. The nucleus was visualized by Hoechst staining. The panels show one representative experiment of four. Bars, 5 µm. (C and D) Neutrophils were cultured for 6 h at 37°C alone or with 50 µM carboxyp21 or modified carboxyp21. The percentages of apoptotic neutrophils were assessed as depolarized mitochondria after DiOC₆ labeling (C) or phosphatidylserine externalization after annexin-V labeling (D). Basal apoptosis was assessed before incubation (fresh). Data are means ± SEM of five independent experiments (*, P < 0.05; **, P < 0.01; Student’s t test). (E) Neutrophils were exposed to increasing carboxyp21 concentrations for 3 h, and apoptosis was measured by annexin-V labeling. The PCNA expression in the same samples was evaluated by Western blot analysis, and the bands were quantified by densitometric scanning. Data are from one representative experiment of four. AU, arbitrary unit. (F) Neutrophils were incubated with or without 1,000 U/ml G-CSF in the presence or absence of 50 µM carboxyp21 for 15 h before determining the percentage of apoptotic cells by annexin-V labeling. Data are means ± SEM of five independent experiments (*, P < 0.05; **, P < 0.01; Student’s t test).