Figure 8.
Figure 8. Disruption of MT network formation inhibits degranulation and DNA release and is regulated by ROS-dependent glutathionylation. Human CGD neutrophils demonstrate defects in tubulin network formation that can be restored by addition of exogenous H2O2. (A) Confocal microscopy. MT assembly was analyzed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors: 1 µM DPI, 1.25 µM Lat B, 1 µM taxol, 5 µM nocodazole, and triggers. Right: Quantification of MT network formation was performed by automated analysis of microscopic images by using Imaris software. n = 5. (B) Confocal microscopy. DNA release was analyzed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors: 1 µM taxol, 5 µM nocodazole, and triggers. Right: Quantification of released dsDNA in supernatants of activated neutrophils. n = 5. (C) Flow cytometry. Expression of CD63, CD66b, and CD35 was assessed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors: 1 µM taxol, 5 µM nocodazole, and triggers. Representative histograms are presented. n = 5. (D) Confocal microscopy. MT assembly was analyzed after short-term stimulation (total 35 min) of control and CGD human neutrophils in the presence or absence of 50 µM H2O2 with the indicated triggers. Quantification of MT network formation was performed by automated analysis of microscopic images by using Imaris software. n = 3. (E) Confocal microscopy. MT assembly was analyzed after short-term stimulation (total 35 min) of WT, Nox2−/−, Grx1−/−, and Was−/− mouse neutrophils with the indicated triggers. Right: Quantification of MT network formation was performed by automated analysis of microscopic images by using Imaris software. n = 5. (F) Glutathionylated proteins in activated human neutrophil lysates were immunoprecipitated with anti–GSH antibody (time-dependent after C5a stimulation) and immunoblotted for tubulin. A total cell lysate (Input) was used as control. Representative data of three independent experiments are shown. Data are means ± SEM. *, P < 0.0231; ***, P < 0.001. Bars, 10 µm.

Disruption of MT network formation inhibits degranulation and DNA release and is regulated by ROS-dependent glutathionylation. Human CGD neutrophils demonstrate defects in tubulin network formation that can be restored by addition of exogenous H2O2. (A) Confocal microscopy. MT assembly was analyzed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors: 1 µM DPI, 1.25 µM Lat B, 1 µM taxol, 5 µM nocodazole, and triggers. Right: Quantification of MT network formation was performed by automated analysis of microscopic images by using Imaris software. n = 5. (B) Confocal microscopy. DNA release was analyzed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors: 1 µM taxol, 5 µM nocodazole, and triggers. Right: Quantification of released dsDNA in supernatants of activated neutrophils. n = 5. (C) Flow cytometry. Expression of CD63, CD66b, and CD35 was assessed after pretreatment and short-term stimulation (total 35 min) of human neutrophils with the indicated inhibitors: 1 µM taxol, 5 µM nocodazole, and triggers. Representative histograms are presented. n = 5. (D) Confocal microscopy. MT assembly was analyzed after short-term stimulation (total 35 min) of control and CGD human neutrophils in the presence or absence of 50 µM H2O2 with the indicated triggers. Quantification of MT network formation was performed by automated analysis of microscopic images by using Imaris software. n = 3. (E) Confocal microscopy. MT assembly was analyzed after short-term stimulation (total 35 min) of WT, Nox2−/−, Grx1−/−, and Was−/− mouse neutrophils with the indicated triggers. Right: Quantification of MT network formation was performed by automated analysis of microscopic images by using Imaris software. n = 5. (F) Glutathionylated proteins in activated human neutrophil lysates were immunoprecipitated with anti–GSH antibody (time-dependent after C5a stimulation) and immunoblotted for tubulin. A total cell lysate (Input) was used as control. Representative data of three independent experiments are shown. Data are means ± SEM. *, P < 0.0231; ***, P < 0.001. Bars, 10 µm.

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