(a) RTPCR analysis of expression of InsP6K isoforms in neutrophils. RNA extracted from brain was used as positive control. The results shown are representative of three experiments. (b) Immunoblot analysis of InsP6K1 protein expression of InsP6K1^−/− and wild-type mouse neutrophils. The results shown are representative of three experiments. (c) Immunoblot analysis of total and phosphorylated Akt in neutrophils stimulated with 1μM fMLP for the indicated time periods. The results shown are representative of three experiments. (d) Ratios of phosphorylated Akt and total Akt in neutrophils stimulated with 1μM fMLP as quantified with NIH Image software²¹. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test). (e) Time-lapse images of murine bone marrow derived neutrophils isolated from InsP6K1^−/− and wild-type mice and transfected with an Akt-PHGFP construct. Cells were stimulated with 1 μM fMLP for the indicated time. Representative fluorescence images of three experiments are shown. (f) Quantitative analysis of membrane translocation of Akt-PH-EGFP in murine bone marrow derived neutrophils stimulated with 1 μM fMLP. The average membrane fluorescence intensities (arbitrary unit, AU) were measured with ImageJ software¹⁶. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test).

(a) Extracellular ROS production monitored by an isoluminol chemiluminescence assay. Bone marrow-derived neutrophils isolated from wild-type and InsP6K1-deficient mice were stimulated with indicated concentration of fMLP, C5a or PMA. Shown are arbitrary light units (ALU) of each measurement. (b) Extracellular ROS production elicited with indicated amount of fMLP. (c) Total amount of ROS production monitored by an cytochrome C reduction assay. Bone marrow neutrophils from wild-type and InsP6K1-deficient mice were incubated with cytochrome-c with or without SOD. Absorbance at 550 nm was measured 5 min after fMLP stimulation. Total amount of ROS production was calculated as the difference in absorbance between samples with and without SOD (ΔA₅₅₀). (d) Intracellular ROS production monitored by an luminol chemiluminescence assay. Bone marrow-derived neutrophils isolated from wild-type and InsP6K1-deficient mice were stimulated with indicated amount of fMLP, C5a or PMA. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test).

(a) Immunoblot analysis of total and phosphorylated Akt in TNP (10μM)-treated and untreated human neutrophils stimulated with 1μM fMLP. The results shown are representative of three experiments. Relative amounts of phosphorylated Akt were quantified with NIH Image software. (b) fMLP-elicited extracellular ROS production in DMSO and TNP-treated human neutrophils. (c) fMLP-elicited intracellular ROS production in human neutrophils. (d) C5a -elicited extracellular ROS production in human neutrophils. (e) C5a-elicited intracellular ROS production in human neutrophils. Data are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test).

(a) PtdIns(3,4,5)P3 levels in TNP (10μM)-treated and untreated human neutrophils unstimulated (us) or stimulated with 1μM fMLP for 2 min. (b) PtdIns(3,4,5)P3 levels in neutrophil-like differentiated HL60 cells (dHL60) unstimulated (us) or stimulated with 1μM fMLP for 2 min. (c) PtdIns(3,4,5)P3 levels in InsP6K1^−/− and wild-type murine neutrophils unstimulated (us) or stimulated with 1μM fMLP for 2 min. Data shown are means ± SD of 3 experiments.

(a) Expression of Myc-tagged InsP6K1, InsP6K2, InsP6K3, and the kinase dead form InsP6K1 (InsP6K1–KD) mutant in dHL60 cells transfected with indicated construct. The blot shown is representative of three experiments. (b) The amounts of InsP7 in cells transfected with indicated constructs as analyzed by HLPC analysis. Inositol phosphates were identified by their co-elution with the standards of H³-inositol phosphates. The data were normalized to the total amount of protein extracted from the same samples. Shown are the peaks of InsP7. The results shown are representative of three experiments. (c) Immunoblot analysis of total and phosphorylated Akt in dHL60 cells unstimulated (us) or stimulated with 1μM fMLP for 3 min. The results shown are representative of three experiments. Relative amounts of phosphorylated Akt were quantified using NIH Image. Data are mean ± SD of 3 independent experiments. (d) fMLP-elicited membrane translocation of Akt-PH-GFP in dHL60 cells over-expressing InsP6Ks. HL60 cells were co-transfected with the Akt-PH-GFP construct and indicated construct. The results shown are representative of three experiments. (e) The average membrane fluorescence intensities in Fig 5c as measured with ImageJ software. Data are mean ± SD of 3 independent experiments. (f) fMLP-elicited ROS production in dHL60 cells over-expressing InsP6Ks. dHL60 cells transfected with indicated construct were uniformly stimulated with 1μM fMLP and extracellular ROS production was measured as described in Fig. 2. Data are mean ± SD of 3 independent experiments.

(a) Reconstitution reactions performed using permeabilized PMN cores and mock-depleted cytosol supplied with ATP, creatine kinase, PMA and NADPH in the presence or absence of GTPγS or wortmannin. (b) GTPγS-induced superoxide production in the presence of InsP7, InsP6, or InsS6. The reaction mixture were incubated for 10 min at 37°C before the assay. Kinetics of NADPH oxidase activation was monitored by luminol-dependent chemiluminescence. Data are means ± SD of 3 independent experiments. *p < 0.01 (Student's t test).

(a) InsP7 level in dHL60 cells unstimulated (us) or stimulated with 1μM fMLP for 1 min. The data were normalized to the total amount of protein extracted from the same samples. Shown are the peaks of InsP6 and InsP7. (b) InsP7 level in dHL60 cells stimulated with 1μM fMLP for indicated time periods. Data shown are mean ± SD of 3 experiments. (c) Production of InsP7 in TNP-treated dHL60 cells. Differentiated HL60 cells were treated with TNP (10μM) or DMSO at 37 °C for 30 min and stimulated with/without 1μM fMLP for 1min. Data shown are mean ± SD of 3 experiments.

(a) ROS accumulation in the inflamed peritoneal cavity of wild-type and InsP6K-deficient mice as monitored by the cytochrome C reduction assay. Total amount of ROS was calculated as the difference in absorbance between samples with and without SOD (ΔA₅₅₀). (b) In vivo killing of E.coli by wild-type and InsP6K-deficient mice as reflected by bacteria colony forming units. Images of representative culture plates are shown. (c) Total numbers of survived E.coli and E.coli killed relative to the number of recruited neutrophils. (d) In vivo killing of S. aureus by wild-type and InsP6K-deficient mice as reflected by bacteria colony forming units. Images of representative culture plates are shown. (e) Total numbers of survived S. aureus and S. aureus killed relative to the number of recruited neutrophils. Data are means ± SD of 3 independent experiments. *p < 0.01 by Student's t test.

(a) In vitro killing of E coli by wild-type and InsP6K-deficient mouse neutrophils. Diluted aliquots were spread on agar plates and incubated overnight at 37°C. In vitro bacterial killing capabilities were reflected by the decrease of bacteria colony forming units (CFU) after incubation with neutrophils. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test). (b) In vitro killing of S. aureus by wild-type and InsP6K-deficient mouse neutrophils. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test). (c) Superoxide production by wild-type and InsP6K-deficient mouse neutrophils in response to Zymosan or E.coli. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test). (d) Phagocytosis-associated ROS production by TNP-treated (10 µM, 2 hours) and untreated dHL60 cells. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test). (e) In vitro killing of internalized E.coli by wild-type and InsP6K-deficient mouse neutrophils as assessed using a gentamicin protection assay. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test). (f) Phagocytosis fluorescein-conjugated zymosan particles by wild-type and InsP6K-deficient mouse neutrophils. The results shown are representative of three experiments. (g) Phagocytosis index as expressed by the number of bioparticles engulfed by 100 neutrophils. More than 200 neutrophils were counted in each group. Results are the means (±SD) of three independent experiments. *p < 0.01 (Student's t test). (h) Binding index as expressed by the number of bioparticles bound by 100 neutrophils. Results are the means (±SD) of three independent experiments.