Modulation of calpain/calpastatin pathway by treatment with G-CSF/dexamethasone. (A) Changes in the expression of the CAST gene after treatment with G-CSF and dexamethasone in vivo () or in vitro (■) were measured by the light cycler RT-PCR. Results are presented as the mean ± SEM from 3 independent donors. (B) Different expression of calpastatin after administration of G-CSF and dexamethasone. Samples of neutrophils isolated from control donors (□) and donors stimulated with G-CSF and dexamethasone (■) were subjected to SDS-PAGE and analyzed on Western blots, stained with antibodies against calpastatin. Equal amounts of cells (1.5 × 10⁶) were dissolved in sample buffer, and loaded in each lane. After staining with fluorescently labeled secondary antibody, the blots were scanned with Odyssey and analyzed with Licor Odyssey 2.1 software. Signals were normalized for p38 expression. The intensity of the 2 highest bands recognized by the antibody, which represent 2 major forms of calpastatin in neutrophils, was analyzed. Results represent data from 5 different donors (mean ± SEM). *P < .05 (significant difference). A representative blot comparing calpastatin levels in control cells and mobilized neutrophils is presented. (C) Samples of neutrophils incubated in the presence or absence of G-CSF and dexamethasone were taken at the indicated times and analyzed by Western blot. When indicated, CHX (10 μg/mL) was added to prevent new protein synthesis. Results represent data from 5 independent experiments (mean ± SEM). *P < .05 (significant difference). (D) Samples of neutrophils incubated in the presence or absence of calpain inhibitor 3 (CI3, 20 μmol/L) or caspase inhibitor zVAD (20 μmol/L) were taken at the indicated time points and analyzed by Western Blot. Results represent data from 5 independent experiments (mean ± SEM). *P < .05 (significant difference). (E) Inhibition of Calpains prolongs neutrophil viability. Neutrophils were isolated from healthy donors and cultured overnight alone or with the addition of G-CSF (10 ng/mL), CI3 (20 μmol/L), or zVAD (20 μM). When indicated, CHX (10 μg/mL) was added to prevent new protein synthesis. Cells negative for annexin V staining were considered to be alive. Results represent data from 6 independent experiments (mean ± SEM). *P < .05 (significant difference).