by So Youn Kim, Hyun Sik Jun, Paul A. Mead, Brian C

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Neutrophil stress and apoptosis underlie myeloid dysfunction in glycogen storage disease type Ib by So Youn Kim, Hyun Sik Jun, Paul A. Mead, Brian C. Mansfield, and Janice Y. Chou Blood Volume 111(12):5704-5711 June 15, 2008 ©2008 by American Society of Hematology

Increase in the expression of ER chaperones in neutrophils of GSD-Ib mice. Increase in the expression of ER chaperones in neutrophils of GSD-Ib mice. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Hema 3–stained cytospins of peritoneal neutrophils. (B) Western-blot analysis of protein extracts of peritoneal neutrophils using antibodies against gelatinase, Gr-1, β-actin, or GAPDH. Each lane contains 50 μg protein. (C) Western-blot analysis of protein extracts of neutrophils using antibodies against GRP78, GRP170, PDI, or β-actin. Each lane contains 50 μg protein. So Youn Kim et al. Blood 2008;111:5704-5711 ©2008 by American Society of Hematology

GSD-Ib neutrophils display increased rate of apoptosis. GSD-Ib neutrophils display increased rate of apoptosis. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative flow cytometric analysis of neutrophil annexin V (x-axis) binding and PI (y-axis) uptake. Numbers on the plots are percentages of total cells. (B) Quantification of annexin V–positive neutrophils determined by immunofluorescence staining. (C) Quantification of caspase-3–positive neutrophils determined by immunofluorescence staining. (D) The DEVD-cleaving activity of active caspase-3 in protein extracts of peritoneal neutrophils. Data represent the means (± SEM) of 3 independent experiments. **P < .001. So Youn Kim et al. Blood 2008;111:5704-5711 ©2008 by American Society of Hematology

GSD-Ib neutrophils exhibit oxidative stress. GSD-Ib neutrophils exhibit oxidative stress. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative flow cytometric analysis of neutrophil carboxy-DCF staining. (B) Quantification of carboxy-DCF–positive neutrophils determined by immunofluorescence staining. Data represent the means (± SEM) of 3 independent experiments. ***P < .001. (C) Oxyblot analysis of carbonyl groups in oxidative modified proteins. Each lane contains 50 μg protein. The vertical line has been inserted to indicate a repositioned gel lane. (D) Representative flow cytometric analysis of neutrophil R-123 staining. (E) Quantification of R-123–positive neutrophils determined by immunofluorescence staining. Data represent the means (± SEM) of 3 independent experiments. **P < .001. (F) Western blot analysis of protein extracts of neutrophils using antibodies against Mn-SOD or β-actin. Each lane contains 50 μg protein. So Youn Kim et al. Blood 2008;111:5704-5711 ©2008 by American Society of Hematology

Bax activation in GSD-Ib neutrophils. Bax activation in GSD-Ib neutrophils. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative immunofluorescence of Bax staining (green fluorescence), Hoechst 33342 nuclei staining (blue fluorescence), and MitoTracker Red mitochondrial staining (red fluorescence), magnification, ×1000, and quantification of Bax-positive neutrophils in unaffected and GSD-Ib mice. Data represent the means (± SEM) of 3 independent experiments. ***P < .001. (B) Western blot analysis of protein extracts of neutrophils using antibodies against Bax, β-actin, or GAPDH. Each lane contains 50 μg protein. So Youn Kim et al. Blood 2008;111:5704-5711 ©2008 by American Society of Hematology

Increased accumulation and release of proapoptotic factors in GSD-Ib neutrophils. Increased accumulation and release of proapoptotic factors in GSD-Ib neutrophils. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Representative immunofluorescence of Smac/Diablo staining (green fluorescence), Hoechst 33342 nuclei staining (blue fluorescence), and MitoTracker Red mitochondrial staining (red fluorescence), magnification, ×1000, and quantification of Smac/Diablo-positive neutrophils in unaffected and GSD-Ib mice. (B) Western-blot analysis of protein extracts of neutrophils using antibodies against Smac/Diablo, β-actin or GAPDH. Each lane contains 50 μg protein. (C) Representative immunofluorescence of Omi/HtrA2 staining (green fluorescence), Hoechst 33 342 nuclei staining (blue fluorescence), and MitoTracker Red mitochondrial staining (red fluorescence), magnification, ×1000, and quantification of Omi/HtrA2-positive neutrophils in unaffected and GSD-Ib mice. Data represent the means (± SEM) of 3 independent experiments. ***P < .001. (D) Western blot analysis of protein extracts of neutrophils using antibodies against Omi/HtrA2, β-actin, or GAPDH. Each lane contains 50 μg protein. So Youn Kim et al. Blood 2008;111:5704-5711 ©2008 by American Society of Hematology

Caspase-9 activation in GSD-Ib neutrophils. Caspase-9 activation in GSD-Ib neutrophils. Peritoneal neutrophils were isolated from 6- to 7-week-old unaffected (+/+) and GSD-Ib (−/−) mice during thioglycollate-elicited peritonitis. (A) Immunofluorescence of active caspase-9 staining (green fluorescence) and Hoechst 33342 nuclei staining (blue fluorescence), magnification ×400. (B) Quantification of caspase-9–positive neutrophils in unaffected and GSD-Ib mice. Data represent the means (± SEM) of 3 independent experiments. ***P < .001. So Youn Kim et al. Blood 2008;111:5704-5711 ©2008 by American Society of Hematology