Figure 2. PCNA is degraded by the... | Rockefeller University Press

Figure 2.

$Figure 2. PCNA is degraded by the proteasome during neutrophil apoptosis. (A) PCNA expression kinetics in neutrophils incubated at 37°C for 1, 3, or 15 h alone (constitutive apoptosis), with 10 ng/ml anti-Fas mAb, or with 0.1 µg/ml gliotoxin. Western blot analysis of neutrophil cytosolic fractions (50 µg/lane) using the anti-PCNA PC10 mAb. β-Actin immunoblotting served as a loading control on the same membrane. (B) Flow cytometric measurement of phosphatidylserine externalization on neutrophils cultured as in A and labeled with annexin-V–FITC and 7-AAD to assess apoptosis and necrosis, respectively. (C and D) Spectrophotometric determination of caspase-8 (C) and caspase-3 (D) activities in neutrophil lysates using their respective specific chromogenic IETD-pNA and DEVD-pNA substrates. (E) Effect of the proteasome inhibitor PS-341 at the indicated concentrations on survival (top) and on PCNA expression in neutrophils (bottom) cultured as in A. Apoptosis was obtained by incubating neutrophils at 37°C for 15 h, and neutrophil survival was evaluated as the percentage of annexin-V− 7-AAD− neutrophils, to exclude cell apoptosis and necrosis, and compared with freshly isolated neutrophils (fresh). Values are means ± SEM of eight independent experiments performed in duplicate (*, P < 0.05; **, P < 0.01; Student’s t test). A representative PCNA immunoblot obtained under the same experimental conditions is shown on the bottom. (F) Effect of 1 µM PS-341 on PCNA expression in neutrophils incubated for 6 h at 37°C with anti-Fas or gliotoxin and compared with freshly isolated neutrophils. The percentages of annexin-V− 7-AAD− neutrophils used to evaluate neutrophil survival were 60 and 85% in anti-Fas–treated neutrophils (without and with PS-341) and 55 and 80% in the gliotoxin-treated neutrophils (without and with PS-341), compared with 99% in untreated neutrophils. The bottom panel shows colloidal gold staining of the membrane used as a loading control. A–D and F show results from representative experiments that were performed at least four times and yielded identical results.$

PCNA is degraded by the proteasome during neutrophil apoptosis. (A) PCNA expression kinetics in neutrophils incubated at 37°C for 1, 3, or 15 h alone (constitutive apoptosis), with 10 ng/ml anti-Fas mAb, or with 0.1 µg/ml gliotoxin. Western blot analysis of neutrophil cytosolic fractions (50 µg/lane) using the anti-PCNA PC10 mAb. β-Actin immunoblotting served as a loading control on the same membrane. (B) Flow cytometric measurement of phosphatidylserine externalization on neutrophils cultured as in A and labeled with annexin-V–FITC and 7-AAD to assess apoptosis and necrosis, respectively. (C and D) Spectrophotometric determination of caspase-8 (C) and caspase-3 (D) activities in neutrophil lysates using their respective specific chromogenic IETD-pNA and DEVD-pNA substrates. (E) Effect of the proteasome inhibitor PS-341 at the indicated concentrations on survival (top) and on PCNA expression in neutrophils (bottom) cultured as in A. Apoptosis was obtained by incubating neutrophils at 37°C for 15 h, and neutrophil survival was evaluated as the percentage of annexin-V⁻ 7-AAD⁻ neutrophils, to exclude cell apoptosis and necrosis, and compared with freshly isolated neutrophils (fresh). Values are means ± SEM of eight independent experiments performed in duplicate (*, P < 0.05; **, P < 0.01; Student’s t test). A representative PCNA immunoblot obtained under the same experimental conditions is shown on the bottom. (F) Effect of 1 µM PS-341 on PCNA expression in neutrophils incubated for 6 h at 37°C with anti-Fas or gliotoxin and compared with freshly isolated neutrophils. The percentages of annexin-V⁻ 7-AAD⁻ neutrophils used to evaluate neutrophil survival were 60 and 85% in anti-Fas–treated neutrophils (without and with PS-341) and 55 and 80% in the gliotoxin-treated neutrophils (without and with PS-341), compared with 99% in untreated neutrophils. The bottom panel shows colloidal gold staining of the membrane used as a loading control. A–D and F show results from representative experiments that were performed at least four times and yielded identical results.

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