Conditioned neutrophil viability is reversible upon reoxygenation and can be modulated by targeting PCNA or Bax expression. (A) Mcl-1, PCNA, and pro-caspase-3 stability was assessed by western blot on freshly purified and conditioned/unconditioned neutrophils (20 hours). Neutrophil viability was quantified by flow cytometry. Error bars indicate SD. **P < .01; “ns” indicates P > .05 (n = 5). (B) Conditioned neutrophils were subsequently exposed to 21% O₂ for 6 and 20 hours. Mcl-1, PCNA, and pro-caspase-3 were detected by western blot, and neutrophil viability was assessed by flow cytometry. Error bars indicate SD. ***P < .001; “ns” indicates P > .05 (n = 5). (C-E) Conditioned neutrophils were transfected with negative control (Neg. Ctrl.) siRNA, PCNA siRNA (C), or Bax siRNA (D); Mcl-1, PCNA, and pro-caspase-3 stability was analyzed by western blot 20 hours (C) or 48 hours (D-E) postnucleofection (after nucleofection, neutrophils were cultured in RMPI supplemented with 10 mM HEPES and 3 mM glucose for the indicated period of time). Neutrophil viability was quantified by flow cytometry (C, E). Error bars indicate SD. ***P < .001; **P < .01; “ns” indicates P > .05 (n = 5).