Journal: Blood

Article Title: Pretreatment with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670 augments the efficacy of granulocyte transfusion in a clinically relevant mouse model

doi: 10.1182/blood-2010-09-309864

Figure Lengend Snippet: PTEN inhibitor SF1670 enhances fMLP-induced PtdIns(3,4,5)P3 signaling in both human and mouse neutrophils. (A) Human primary neutrophils were pretreated with 500nM SF1670 at 37°C for 30 minutes and then stimulated with 100nM fMLP for indicated times. The levels of phosphorylated AKT, p38, and c-Jun NH2-terminal kinase (JNK) were assessed by Western blotting with specific antibodies. (B) Human neutrophils were incubated with indicated concentrations of SF1670 at 37°C for 30 minutes and then stimulated with or without 100nM fMLP for 4 minutes. Total and phosphorylated AKT levels were assessed as described in panel A. (C) Human neutrophils were incubated with 500nM SF1670 at 37°C for 30 minutes and then washed twice with PBS. After being incubated in PBS + 2% BSA for indicated times, cells were stimulated with 100nM fMLP for 4 minutes, and then the levels of total and phosphorylated AKT were assessed by Western blotting using specific antibodies. (D) PTEN inhibitor SF1670 enhanced fMLP-induced PtdIns(3,4,5)P3 signaling in mouse neutrophils. Mouse bone marrow derived neutrophils were purified by negative selection using an EasySep system (StemCell Technologies). Cells were pretreated with 500nM SF1670 at 37°C for 30 minutes and then stimulated with 500nM fMLP for indicated times. The levels of phosphorylated AKT were assessed by Western blotting with specific antibody. (E) PtdIns(3,4,5)P3 level increased in SF1670-treated mouse neutrophils. Cellular PtdIns(3,4,5)P3 levels were measured using a mass ELISA kit (Echelon). In brief, PtdIns(3,4,5)P3 was extracted from 5 million purified mouse bone marrow–derived neutrophils following the manufacturer's lipid extraction protocol. The samples were mixed and incubated with a PtdIns(3,4,5)P3 detector protein and then added to a PtdIns(3,4,5)P3-coated microplate for competitive binding. A peroxidase-linked secondary detector and colorimetric detection were used to quantify the PtdIns(3,4,5)P3 detector protein bound to the plate. The colorimetric signal is inversely proportional to the amount of PtdIns(3,4,5)P3 in the sample. (F) Bone marrow–derived neutrophils isolated from wild-type (WT) and myeloid specific PTEN knockout mice28 were preincubated with or without 500nM SF1670 at 37°C for 30 minutes. Cells were then stimulated with or without 1 μM of fMLP for 4 minutes. Total and phosphorylated AKT levels were measured as described in panel A.

Article Snippet: Cellular PtdIns(3,4,5)P3 levels were measured using a mass ELISA kit (Echelon).

Techniques: Western Blot, Incubation, Derivative Assay, Purification, Selection, Enzyme-linked Immunosorbent Assay, Binding Assay, Isolation, Knock-Out

Journal: Blood

Article Title: Pretreatment with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670 augments the efficacy of granulocyte transfusion in a clinically relevant mouse model

doi: 10.1182/blood-2010-09-309864

Figure Lengend Snippet: Pretreatment with SF1670 enhances the function and increases the efficacy of transfusion of G-CSF–mobilized neutrophils. (A) G-CSF (GCSF) induced neutrophil mobilization. A wild-type C57Bl/6 mouse was subcutaneously injected with G-CSF once daily at 250 μg/kg for 5 days. Peripheral blood (PB) was collected by cardiopuncture. White blood cells were counted using a Hematology Analyzer (Hemavet). (B) PTEN inhibitor SF1670 (SF) enhances fMLP-induced PtdIns(3,4,5)P3 signaling in both G-CSF–mobilized peripheral blood neutrophils and bone marrow–derived neutrophils (BM). Mouse peripheral blood samples were mixed with 2% dextran sulfate/PBS and 3 mg/mL EDTA/PBS at the ratio of 1:1:1 and incubated at 37°C for 20 minutes to allow red blood cells to settle. The leukocyte-containing layer was removed, and neutrophils were isolated by negative selection as described here. Isolated neutrophils were treated with SF1670 (500nM) or DMSO at 37°C for 30 minutes in PBS (0.2% bovine serum albumin), washed twice with HBSS, and then stimulated with 500nM fMLP for 4 minutes. The levels of phosphorylated AKT were assessed by Western blotting with specific antibody. (C) PTEN inhibitor SF1670 enhances fMLP-induced ROS production in G-CSF–mobilized peripheral blood neutrophils. Neutrophils were treated with SF1670 (500nM) or DMSO at 37°C for 30 minutes in PBS (0.2% BSA), washed twice with HBSS, and then stimulated with 2μM fMLP. ROS production was monitored in the presence of 50μM isoluminol and 0.8 U of horseradish peroxidase in a luminometer at 37°C. Chemiluminescence (arbitrary light units) was recorded at indicated time points. (D) Pretreatment with SF1670 increases the efficacy of transfusion of G-CSF–mobilized neutrophils. G-CSF–mobilized mouse peripheral blood neutrophils were pretreated with DMSO or SF1670 (250nM) for 30 minutes at 37°C, washed twice with PBS, and then intravenously injected into neutropenic mice that had been challenged with 105 CFU of E coli for 5 hours. Mice not receiving granulocyte transfusion were used as control. Histologic analysis of lungs reveals bacterial colonies (white arrow) in the pulmonary parenchyma. The number of bacterial colony was calculated. Pulmonary edema formation was quantified as the percentage of edema in the total parenchymal region using IPLab imaging software as described previously.29. *P < .05, **P < .01 vs control; ##P < .01 vs DMSO.

Article Snippet: Cellular PtdIns(3,4,5)P3 levels were measured using a mass ELISA kit (Echelon).

Techniques: Injection, Derivative Assay, Incubation, Isolation, Selection, Western Blot, Imaging, Software