Treatment with PTEN inhibitor SF1670 augments fMLP-induced polarization and chemotaxis of mouse neutrophils. (A-C) fMLP induced neutrophil polarization. Bone marrow–derived mouse neutrophils were pretreated with 250nM SF1670 at 37°C for 30 minutes, washed twice with HBSS, and then settled down for 5 minutes on LabTek chamber cover-glass coated with 30 μg/mL bovine fibronectin. Cell polarization was induced by uniform stimulation with indicated concentrations of fMLP. (A) Representative images of cell ruffling (5 minutes after fMLP stimulation). (B) Quantification of fMLP-induced cell ruffling. The percentage of neutrophils extending pseudopods or ruffling was calculated from fields captured 4 to 8 minutes after fMLP stimulation. (C) Cells were pretreated with DMSO (control) or indicated concentrations of SF1670 at 37°C for 30 minutes and then stimulated with 25nM fMLP. The percentage of neutrophils extending pseudopods or ruffling was calculated from fields captured 4 to 8 minutes after fMLP stimulation. (D-G) Chemoattractant-induced transwell chemotaxis of neutrophils. Bone marrow–derived mouse neutrophils were pretreated with indicated amount of SF1670 at 37°C for 30 minutes, washed twice with HBSS, and then allowed to migrate in response to the indicated chemoattractants: fMLP (D), C5a (E), leukotriene B4 (LTB4; F), and macrophage-inflammatory protein-2 (G) in transwell systems. Percentage of cells that migrated into the bottom well was recorded. Data shown are mean ± SD of 6 wells, from 1 experiment representative of 3 experiments.