1. G-CSF improves murine G6PC3-deficient neutrophil function by modulating apoptosis and energy homeostasis
by Hyun Sik Jun, Young Mok Lee, Ki Duk Song, Brian C. Mansfield, and Janice Y. Chou
Blood
Volume 117(14):
April 7, 2011
©2011 by American Society of Hematology

2. The ER stress and cell survival signaling pathways in G6pc3−/− BM neutrophils.
The ER stress and cell survival signaling pathways in G6pc3−/− BM neutrophils. BM neutrophils were isolated from 6- to 8-week-old control (+/+) and G6pc3−/− (−/−) littermates as described in “Neutrophil isolation.” (A) Western blot analysis of protein extracts of neutrophils using antibodies against p-PERK, eIF2α, p-eIF2α, ATF4, CHOP, or β-actin. Each lane contains 50 μg protein. The relative protein levels of p-PERK, p-eIF2α, ATF4, or CHOP were quantified by densitometry of 3 or 4 separate pairs of Western blots. (B) Western blot analysis of protein extracts of neutrophils using antibodies against p-Akt, total Akt, p-SHIP1, SHIP1, PI3K-p85α, PI3K-p110α PTEN, p-PTEN, or β-actin. Each lane contains 50 μg protein. The relative protein levels of p-Akt or p-SHIP1 were quantified by densitometry of 3 or 4 separate pairs of Western blots. (C-D) Flow cytometric analysis of p-Akt (C) or PtdIns(3,4,5)P3 (D). Data are the mean ± SEM of 4 independent experiments. **P < *P < .05.
Hyun Sik Jun et al. Blood 2011;117:
©2011 by American Society of Hematology

3. Oxidative stress and intrinsic mitochondrial apoptotic pathway in G6pc3−/− neutrophils.
Oxidative stress and intrinsic mitochondrial apoptotic pathway in G6pc3−/− neutrophils. BM neutrophils were isolated from 6- to 8-week-old control (+/+) and G6pc3−/− (−/−) littermates as described in “Neutrophil isolation.” (A) Quantitative flow cytometric analysis of neutrophil carboxy-DCF staining. Data are the mean ± SEM of 4 independent experiments. (B) Western blot analysis of protein extracts of neutrophils using antibodies against Mn-SOD or β-actin. Each lane contains 50 μg protein. The relative protein levels of Mn-SOD were quantified by densitometry of 3 separate pairs of Western blots. (C) Representative confocal microscopic analysis of Bax (green fluorescence), COX IV (red fluorescence, mitochondria), and DAPI nuclei (blue fluorescence) staining (original magnification ×1000). (D) Western blot analysis of protein extracts of neutrophils using antibodies against Bax, Smac/Diablo, Omi/HtrA2, or β-actin. Each lane contains 50 μg protein. The relative protein levels of Bax, Smac/Diablo, or Omi/HtrA2 were quantified by densitometry of 3 or 4 separate pairs of Western blots. (E) Quantitative flow cytometric analysis of cytochrome c release. Data are the mean ± SEM of 3 independent experiments. (F) Flow cytometry, immunoprecipitation, and Western blot analysis of immunoprecipitates using an antibody against active caspase-9 and a horseradish peroxidase-conjugated secondary antibody. Data for flow cytometric analysis represent the mean ± SEM of 4 independent experiments. (G) Quantitative flow cytometric analysis of active caspase-3. Data are the mean ± SEM of 4 independent experiments. **P < *P < .05.
Hyun Sik Jun et al. Blood 2011;117:
©2011 by American Society of Hematology

4. G-CSF can delay but not prevent apoptotic death of G6pc3−/− BM neutrophils cultured in vitro.
G-CSF can delay but not prevent apoptotic death of G6pc3−/− BM neutrophils cultured in vitro. The annexin V–depleted BM neutrophils were isolated from 6- to 8-week-old unaffected (+/+, ○, ●) and G6pc3−/− (−/−, Δ, ▴) littermates as described in “Neutrophil isolation.” (A) Neutrophil viability. (B) Representative flow cytometric analysis of neutrophil survival. (C) Quantitative analysis of neutrophil survival in untreated wild-type (○, ●) and G6pc3−/− (Δ, ▴) mice in the absence (○, Δ) or presence (●, ▴) of G-CSF. Data are the mean ± SEM of 4 independent experiments. (D) Flow cytometric analysis of G-CSFR. (E) Western blot analysis of protein extracts of neutrophils using antibodies against GRP78, PDI, Bax, Smac/Diablo, or β-actin. Each lane contains 50 μg protein. (F) Quantitative flow cytometric analysis of p-Akt in neutrophils of untreated wild-type (○, ●) and G6pc3−/− (Δ, ▴) mice in the absence (○, Δ) or presence (●, ▴) of G-CSF. Data are the mean ± SEM of 3 independent experiments. **P < *P < .05.
Hyun Sik Jun et al. Blood 2011;117:
©2011 by American Society of Hematology

5. Effects of limiting glucose, fMLP, C5a, CytB, and 2-DG on apoptotic death of G6pc3−/− BM neutrophils cultured in vitro.
Effects of limiting glucose, fMLP, C5a, CytB, and 2-DG on apoptotic death of G6pc3−/− BM neutrophils cultured in vitro. The annexin V–depleted BM neutrophils were isolated from 6- to 8-week-old unaffected (+/+) and G6pc3−/− (−/−) littermates as described in “Neutrophil isolation.” (A) Quantitative analysis of neutrophil survival in untreated wild-type (○, ●, □, ■) and G6pc3−/− (Δ, ▴, ▿, ▾) mice in 6.2mM glucose (○, ●, Δ, ▴,) or 0.6mM glucose (□, ■, ▿, ▾)-containing medium in the absence (○, □, Δ, ▿) or presence (●, ■, ▴, ▾) of G-CSF. Data are the mean ± SEM of 4 independent experiments. (B) Effect of fMLP, C5a, or CytB on neutrophil 2-DG uptake. Glucose uptake was examined in annexin V–depleted BM neutrophils that were treated at 37°C for 15 minutes with 10−6M fMLP, 0.8 μg/mL C5a, or 5 μg/mL CytB. Data are the mean ± SEM of 3 independent experiments. (C) Quantitative analysis of neutrophil survival in untreated wild-type (○, ×, □, ●) and G6pc3−/− (Δ, ▿, ★, ▴) mice in 6.2mM glucose-containing medium in the presence of 10−6M fMLP (×, ▿), 0.8 μg/mL C5a (□, ★), or 5 μg/mL CytB (●, ▴). (D) Quantitative analysis of neutrophil survival in untreated wild-type (○, ●, □, ■) and G6pc3−/− (Δ, ▴, ▿, ▾) mice in 6.2mM glucose-containing medium in the absence (○, Δ, ●, ▴) or presence of 1mM 2-DG (□, ■, ▿, ▾), in the presence of G-CSF (●, ▴), or 2-DG/G-CSF (■, ▾). **P < *P < .05.
Hyun Sik Jun et al. Blood 2011;117:
©2011 by American Society of Hematology

6. G-CSF delays G6pc3−/− neutrophil apoptosis by modulating apoptotic mediators.
G-CSF delays G6pc3−/− neutrophil apoptosis by modulating apoptotic mediators. The annexin V–depleted BM neutrophils were isolated from 6- to 8-week-old unaffected (+/+, ○, ●) and G6pc3−/− (−/−, Δ, ▴) littermates as described in “Neutrophil isolation.” (A) Western blot analysis of protein extracts of neutrophils using antibodies against Mcl-1, Bax, cleaved PARP, total GSK-3β, p-GSK-3β (Ser9), or β-actin. Each lane contains 50 μg protein. The relative protein levels of Mcl-1, Bax, cleaved PARP, total GSK-3β, or p-GSK-3β (Ser9) were quantified by densitometry of 3 or 4 separate pairs of Western blots. (B) Representative confocal microscopic analysis of Bax (green fluorescence), COX IV (red fluorescence, mitochondria), and DAPI nuclei (blue fluorescence) staining (original magnification ×1000). (C) Quantitative flow cytometric analysis of active caspase-9 and caspase-3 in neutrophils of untreated wild-type (○, ●) and G6pc3−/− (Δ, ▴) mice in the absence (○, Δ) or presence (●, ▴) of G-CSF. Data are the mean ± SEM of 3 independent experiments. **P < *P < .05.
Hyun Sik Jun et al. Blood 2011;117:
©2011 by American Society of Hematology

7. In vivo G-CSF therapy corrects neutropenia and improves neutrophil function in G6pc3−/− mice.
In vivo G-CSF therapy corrects neutropenia and improves neutrophil function in G6pc3−/− mice. G6pc3−/− (−/−) and control (+/+) littermates were treated with recombinant mouse G-CSF for 1 or 5 days. (A) Blood and BM neutrophil counts in untreated and G-CSF–treated mice. (B) Representative flow cytometric analysis of neutrophil p-Akt, membrane-bound PtdIns(3,4,5)P3, and active caspase-3 in 5-day G-CSF–treated control and G6pc3−/− mice. (C) Neutrophil respiratory burst activity in response to 200 ng/mL of phorbol myristate acetate in untreated and 5-day G-CSF–treated control (○) and G6pc3−/− (●) mice. (D) Neutrophil concentration-dependent chemotaxis in response to fMLP in untreated and 5-day G-CSF–treated control (○) and G6pc3−/− (●) mice. (E) Neutrophil calcium flux in response to 10−6M of fMLP in untreated and 5-day G-CSF–treated control (○) and G6pc3−/− (●) mice. Data are the mean ± SEM of 3 (untreated) or 6 (G-CSF–treated) independent experiments. *P < .05.
Hyun Sik Jun et al. Blood 2011;117:
©2011 by American Society of Hematology

8. In vivo G-CSF therapy increases neutrophil 2-DG uptake, the expression of GLUT1, and intracellular G6P, lactate, and ATP levels.
In vivo G-CSF therapy increases neutrophil 2-DG uptake, the expression of GLUT1, and intracellular G6P, lactate, and ATP levels. BM neutrophils were isolated from 6- to 8-week-old control (+/+) and G6pc3−/− (−/−) littermates after 5-day G-CSF therapy. (A) Neutrophil 2-DG uptake in untreated and 5-day G-CSF–treated control and G6pc3−/− mice. Data are the mean ± SEM of 3 (untreated) or 6 (G-CSF–treated) independent experiments. (B) Representative immunofluorescence of neutrophil GLUT1 staining (green fluorescence), pan Cadherin membrane staining (red fluorescence), and DAPI nuclei staining (blue fluorescence) (original magnification ×1000). (C) Neutrophil G6P, lactate, and ATP levels in untreated and 5-day G-CSF–treated control and G6pc3−/− mice. Data are the mean ± SEM of 3 (untreated) or 6 (G-CSF–treated) independent experiments. (D) Quantitative flow cytometric analysis of survival of in vitro cultured neutrophils isolated from 5-day G-CSF–treated wild-type (○, ●) and G6pc3−/− (Δ, ▴) mice in the absence (○, Δ) or presence (●, ▴) of G-CSF. Data are the mean ± SEM of 4 independent experiments. **P < *P < .05.
Hyun Sik Jun et al. Blood 2011;117:
©2011 by American Society of Hematology