Analysis of levels of Hsp90, HIF-1α, and PPAR-γ in G6PT-deficient neutrophils and the effects of PPAR-γ antagonist/agonist on neutrophil function. Annexin V–depleted peripheral blood neutrophils isolated from HDs and GSD-Ib patients were used in the study. (A) Quantification of Hsp90 and HIF-1α mRNA levels by real-time RT-PCR and protein levels by densitometry. Data for RT-PCR represent the mean ± SEM for HDs (n = 10) and GSD-Ib patients (n = 12), and data for protein levels represent mean ± SEM for HDs (n = 11) and GSD-Ib patients (n = 7). (B) Western blot analysis of protein extracts using antibodies against Hsp90, HIF-1α, PPAR-γ, or β-actin. Data represent the mean ± SEM for HDs (n = 11) and GSD-Ib patients (n = 7). (C) Immunofluorescence of HIF-1α (green fluorescence) and DAPI nuclei staining (blue fluorescence) at original magnification ×400 and quantification of the relative integrated fluorescence intensity by ImageJ. (D) Quantification of PPAR-γ mRNA levels by real-time RT-PCR and protein levels by densitometry. Data for RT-PCR represent the mean ± SEM for HDs (n = 10) and GSD-Ib patients (n = 12), and data for PPAR-γ protein represent the mean ± SEM for HDs (n = 11) and GSD-Ib patients (n = 7). (E) Western blot analysis of the effects of G-CSF on PPAR-γ expression in HD neutrophils after in vitro culturing. (F) Effects of PPAR-γ agonist rosiglitazone and antagonist GW9662 on function of neutrophils isolated from HDs. Three independent experiments were conducted with similar results. Chemotaxis was examined in response to 10−7 M fMLP. Data represent the mean ± SEM. Calcium mobilization was examined in response to 10−7 M fMLP. Representative profiles are shown. Respiratory burst was examined in response to PMA. Representative profiles are shown. ○, control; ▲, PPAR-γ agonist rosiglitazone; ●, PPAR-γ agonist rosiglitazone followed by antagonist GW9662. (G) Effects of PPAR-γ antagonist GW9662 on function of G6PT-deficient neutrophils isolated from 2 GSD-Ib patients. Two independent experiments using neutrophils isolated from P11 and P14 were conducted. Chemotaxis was examined in response to 10−7 M fMLP. Data represent the mean ± SEM of both patients. Calcium mobilization was examined in response to 10−7 M fMLP. Respiratory burst was examined in response to PMA. ○, control; ●, PPAR-γ antagonist GW9662. **P < .005, *P < .05.
