Figure 6.
Figure 6. Disruption of PTENα attenuates chemoattractant-induced migration and chemotaxis in vivo and in vitro. (A) Absolute number of neutrophils in elicited peritoneal cells without injection or after intraperitoneal injection with thioglycollate. BD Trucount Absolute count tubes were used to count absolute cell numbers. Data represent mean ± SEM (≥6 mice in each group). This result was successfully repeated in 3 independent experiments with 4 or 5 mice per group. **P < .01, 2-way repeated-measures ANOVA, followed by the Bonferroni post test. (B-C) In vitro Transwell chemotaxis. The percentage of neutrophils that migrated into lower wells in response to concentration gradients of fMLP (B) and MIP-2 (C). *P < .05, **P < .01, 2-way repeated-measures ANOVA, followed by the Bonferroni post test. (D-E) Number of neutrophils that invaded the membrane in response to 5 nM fMLP. (D) Invading neutrophils were stained with crystal violet; random invasion control (left panels) and fMLP-stimulated invasion (right panels) are shown. An IX53 Inverted Microscope and Olympus cellSens image-acquisition software (both from Olympus, Tokyo, Japan) were used for image capture (40× lens objective). (E) The percentage of migrating cells was calculated as the number of invading cells divided by the number of seeded cells. Data are representative of 3 independent experiments with a total of 3 or 4 mice per group (mean ± standard error of the mean). *P < .05, 2-way repeated-measures ANOVA, followed by the Bonferroni post test. ns, not significant (P > .05). (F) In vitro under-agarose cell-migration assay. The chemotaxis index is represented by the distance of directional migrated neutrophils. Images are shown in the right panel. WT neutrophils (upper right panels) and PTENα−/− neutrophils (lower right panels) were treated with 1 pM fMLP, and chemotaxis toward fMLP was determined. An IX53 Inverted Microscope and Olympus cellSens image-acquisition software were used for image capture (10× lens objective). Data are representative of 3 independent experiments with a total of 4 or 5 mice per group (mean ± standard error of the mean). ***P < .001, 2-tailed unpaired Student t test.

Disruption of PTENα attenuates chemoattractant-induced migration and chemotaxis in vivo and in vitro. (A) Absolute number of neutrophils in elicited peritoneal cells without injection or after intraperitoneal injection with thioglycollate. BD Trucount Absolute count tubes were used to count absolute cell numbers. Data represent mean ± SEM (≥6 mice in each group). This result was successfully repeated in 3 independent experiments with 4 or 5 mice per group. **P < .01, 2-way repeated-measures ANOVA, followed by the Bonferroni post test. (B-C) In vitro Transwell chemotaxis. The percentage of neutrophils that migrated into lower wells in response to concentration gradients of fMLP (B) and MIP-2 (C). *P < .05, **P < .01, 2-way repeated-measures ANOVA, followed by the Bonferroni post test. (D-E) Number of neutrophils that invaded the membrane in response to 5 nM fMLP. (D) Invading neutrophils were stained with crystal violet; random invasion control (left panels) and fMLP-stimulated invasion (right panels) are shown. An IX53 Inverted Microscope and Olympus cellSens image-acquisition software (both from Olympus, Tokyo, Japan) were used for image capture (40× lens objective). (E) The percentage of migrating cells was calculated as the number of invading cells divided by the number of seeded cells. Data are representative of 3 independent experiments with a total of 3 or 4 mice per group (mean ± standard error of the mean). *P < .05, 2-way repeated-measures ANOVA, followed by the Bonferroni post test. ns, not significant (P > .05). (F) In vitro under-agarose cell-migration assay. The chemotaxis index is represented by the distance of directional migrated neutrophils. Images are shown in the right panel. WT neutrophils (upper right panels) and PTENα−/− neutrophils (lower right panels) were treated with 1 pM fMLP, and chemotaxis toward fMLP was determined. An IX53 Inverted Microscope and Olympus cellSens image-acquisition software were used for image capture (10× lens objective). Data are representative of 3 independent experiments with a total of 4 or 5 mice per group (mean ± standard error of the mean). ***P < .001, 2-tailed unpaired Student t test.

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