Figure 7
Figure 7. Notch signaling is required for GM-CSF–induced eosinophil shape change and chemokinesis. (A) Eosinophils were cultured overnight with increasing doses of GM-CSF in the presence of 10 μM GSI or vehicle alone, fixed with 4% PFO, and analyzed using the FSC parameter of flow cytometry to assess cell shape changes. Data represent average FSC geometric means (± SD) for 1 experiment representative of 8 independent experiments. (i) Live cells were imaged using phase microscopy with an inverted microscope before fixation (imaged with a Nikon TE-300 inverted microscope coupled to a Retiga Exi cooled digital camera; images acquired using QImaging software). (ii) Fixed and permeabilized cells were stained with Alexa 488–conjugated phalloidin to detect F-actin filaments and Hoescht for nuclear staining, as described in “Methods” (imaged with a BX62 Olympus upright microscope, 60× PlanApo objective with numerical aperture of 1.42, coupled to a Hamamatsu Orca AG fire-wire cooled digital camera; images acquired using IPLab). (B) Data from panel A were normalized to the relevant nonstimulated values and replotted. (A,B) Statistical significances between curves were confirmed using a 2-way ANOVA test, followed by Bonferroni posttests to determine significances at individual doses. (C) Eosinophils were stimulated with 100 pM GM-CSF in the presence of 10 μM GSI or vehicle alone for 1, 3, 5, or 8 hours. (D) Eosinophils were cultured overnight in medium alone (top panel) or with 100 pM GM-CSF (bottom panel) in the presence of 10 μM GSI (dotted line) or vehicle alone (solid line) before staining for CD69 surface expression. Data shown are from 1 experiment representative of 3 independent experiments. Shaded histogram represents isotype control. (E,F) Eosinophils were primed for 5 hours with 1 pM GM-CSF in the presence or absence of γ-secretase inhibitors (E) or neutralizing Abs against Notch 1 (F). After pretreatment, cells were added to upper wells of chemotaxis chambers, and chemokinesis within 1 hour was determined by quantifying cells migrating to the lower wells. *P < .05 versus vehicle alone or isotype control–treated samples.

Notch signaling is required for GM-CSF–induced eosinophil shape change and chemokinesis. (A) Eosinophils were cultured overnight with increasing doses of GM-CSF in the presence of 10 μM GSI or vehicle alone, fixed with 4% PFO, and analyzed using the FSC parameter of flow cytometry to assess cell shape changes. Data represent average FSC geometric means (± SD) for 1 experiment representative of 8 independent experiments. (i) Live cells were imaged using phase microscopy with an inverted microscope before fixation (imaged with a Nikon TE-300 inverted microscope coupled to a Retiga Exi cooled digital camera; images acquired using QImaging software). (ii) Fixed and permeabilized cells were stained with Alexa 488–conjugated phalloidin to detect F-actin filaments and Hoescht for nuclear staining, as described in “Methods” (imaged with a BX62 Olympus upright microscope, 60× PlanApo objective with numerical aperture of 1.42, coupled to a Hamamatsu Orca AG fire-wire cooled digital camera; images acquired using IPLab). (B) Data from panel A were normalized to the relevant nonstimulated values and replotted. (A,B) Statistical significances between curves were confirmed using a 2-way ANOVA test, followed by Bonferroni posttests to determine significances at individual doses. (C) Eosinophils were stimulated with 100 pM GM-CSF in the presence of 10 μM GSI or vehicle alone for 1, 3, 5, or 8 hours. (D) Eosinophils were cultured overnight in medium alone (top panel) or with 100 pM GM-CSF (bottom panel) in the presence of 10 μM GSI (dotted line) or vehicle alone (solid line) before staining for CD69 surface expression. Data shown are from 1 experiment representative of 3 independent experiments. Shaded histogram represents isotype control. (E,F) Eosinophils were primed for 5 hours with 1 pM GM-CSF in the presence or absence of γ-secretase inhibitors (E) or neutralizing Abs against Notch 1 (F). After pretreatment, cells were added to upper wells of chemotaxis chambers, and chemokinesis within 1 hour was determined by quantifying cells migrating to the lower wells. *P < .05 versus vehicle alone or isotype control–treated samples.

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