Figure 3
Figure 3. Formation of multiple pseudopodia in PTEN−/− neutrophils. WT and PTEN−/− neutrophils (4 × 105) were plated on glass-bottomed dishes and stimulated with 100 nM fMLP for 5 minutes. Cells were then fixed with 4% formaldehyde, permeabilized with 0.2% Triton-X 100, and stained with fluorescein phalloidin (for F-actin) and Hoechst dye (nuclear stain). (A) Representative DIC/Nomarski and phalloidin (green)/Hoechst (blue) stained images of PTEN−/− neutrophils showing multiple pseudopodia (top). Images were captured using a 96×/1.42 NA oil objective (Olympus). Most WT neutrophils show a single pseudopod (bottom). White arrows indicate pseudopods. (B) Percentage of polarized PTEN−/− and WT neutrophils showing multiple pseudopodia 5 minutes after stimulation with 100 nM fMLP; 50 cells were evaluated for each case. (C-D) F-actin localization in adhered versus suspended PTEN−/− and WT neutrophils. (C) WT and PTEN−/− neutrophils were plated on fibronectin precoated dishes, stimulated with 100 nM fMLP for 5 minutes, fixed with 4% formaldehyde, permeabilized with 0.2% Triton-X-100, and stained with 0.13 mg/mL phalloidin. Images were captured using a 60×/1.42 oil objective (Olympus). WT neutrophils (top, left) show distinct F-actin folds at their leading edges, whereas PTEN−/− neutrophils (bottom, left) are more spread out and do not have as intense F-actin localization at their leading edges. Polarized WT and PTEN−/− neutrophils (n = 51) were scored for clear F-actin localization at the leading edge (with no staining at cell tail or body) and represented in bar graph (right). (D) When neutrophils were stimulated in suspension, the difference in F-actin localization at the leading edge was not apparent. Both WT and PTEN−/− neutrophils intensely localized phalloidin at their leading edges. PTEN−/− neutrophils with multiple pseudopodia are shown in red boxes. Polarized WT and PTEN−/− neutrophils (n = 43) were scored for F-actin localization at their leading edges (with no staining at cell tail or body) and represented in bar graph (right). Images were captured using a 40×/1.35 NA oil objective (Olympus).

Formation of multiple pseudopodia in PTEN−/− neutrophils. WT and PTEN−/− neutrophils (4 × 105) were plated on glass-bottomed dishes and stimulated with 100 nM fMLP for 5 minutes. Cells were then fixed with 4% formaldehyde, permeabilized with 0.2% Triton-X 100, and stained with fluorescein phalloidin (for F-actin) and Hoechst dye (nuclear stain). (A) Representative DIC/Nomarski and phalloidin (green)/Hoechst (blue) stained images of PTEN−/− neutrophils showing multiple pseudopodia (top). Images were captured using a 96×/1.42 NA oil objective (Olympus). Most WT neutrophils show a single pseudopod (bottom). White arrows indicate pseudopods. (B) Percentage of polarized PTEN−/− and WT neutrophils showing multiple pseudopodia 5 minutes after stimulation with 100 nM fMLP; 50 cells were evaluated for each case. (C-D) F-actin localization in adhered versus suspended PTEN−/− and WT neutrophils. (C) WT and PTEN−/− neutrophils were plated on fibronectin precoated dishes, stimulated with 100 nM fMLP for 5 minutes, fixed with 4% formaldehyde, permeabilized with 0.2% Triton-X-100, and stained with 0.13 mg/mL phalloidin. Images were captured using a 60×/1.42 oil objective (Olympus). WT neutrophils (top, left) show distinct F-actin folds at their leading edges, whereas PTEN−/− neutrophils (bottom, left) are more spread out and do not have as intense F-actin localization at their leading edges. Polarized WT and PTEN−/− neutrophils (n = 51) were scored for clear F-actin localization at the leading edge (with no staining at cell tail or body) and represented in bar graph (right). (D) When neutrophils were stimulated in suspension, the difference in F-actin localization at the leading edge was not apparent. Both WT and PTEN−/− neutrophils intensely localized phalloidin at their leading edges. PTEN−/− neutrophils with multiple pseudopodia are shown in red boxes. Polarized WT and PTEN−/− neutrophils (n = 43) were scored for F-actin localization at their leading edges (with no staining at cell tail or body) and represented in bar graph (right). Images were captured using a 40×/1.35 NA oil objective (Olympus).

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