Figure 5
Figure 5. Cdc42/WASp axis controls neutrophil polarity via microtubule orientation and stability. Neutrophils were un-stimulated or stimulated with fMLP on Fg. (A) Immunofluorescence of F-actin and microtubules. Scatter plot for MT polarity as ratio of MT intensity at the back versus the front. Scattered plot representation of at least 90 cells analyzed from 3 independent experiments **P < .0001. Arrow indicates the tail of neutrophils identified on phase contrast images based on the classic morphology of the tail at the uropod (B) Western blots of WCL probed with Glu-tubulin for stabilized microtubules. α-tubulin and p38 were used for total tubulin and loading control (mean ± SD; 3 independent experiments; *P = .0059). (C) Immunofluorescence analysis of EB1 and F-actin. 2D representation of intensity profile of EB1 and F-actin in the region of interest indicated by the box were analyzed in ImageJ Version 1.43J software. Scatter graph for ratio of intensity of EB-1 at back versus front (identified using F-actin). Thirty cells were analyzed from individual experiment and 3 independent experiments were performed (*P = .003). (D) WT neutrophils were transfected with GFP-EB1CΔAC or GFP plasmid vector control using nucleofactor and stimulated with fMLP and Fg and stained for F-actin. Histogram is cells with more than 1 protrusion. Fifty cells were analyzed from individual experiment and 3 independent experiments were performed (mean ± SD; *P = .0027). (E) Immunofluorescence analysis of F-actin and microtubules after stimulation. Scatter plot for MT polarity quantification (mean ± SD; **P = .000017). (F) Western blots of WCL probed with Glu-tubulin (**P = .0024). In fluorescence images tubulin and EB-1 were stained with secondary antibody conjugated with Alexa Fluor 488, while F-actin was labeled with Rhodamine Phalloidin and slides were mounted in SlowFade Gold Antifade. Fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63× objective N/A1.3 with ORCA-ER C4742-95 camera driven by Openlab Version 5.5.0 software.

Cdc42/WASp axis controls neutrophil polarity via microtubule orientation and stability. Neutrophils were un-stimulated or stimulated with fMLP on Fg. (A) Immunofluorescence of F-actin and microtubules. Scatter plot for MT polarity as ratio of MT intensity at the back versus the front. Scattered plot representation of at least 90 cells analyzed from 3 independent experiments **P < .0001. Arrow indicates the tail of neutrophils identified on phase contrast images based on the classic morphology of the tail at the uropod (B) Western blots of WCL probed with Glu-tubulin for stabilized microtubules. α-tubulin and p38 were used for total tubulin and loading control (mean ± SD; 3 independent experiments; *P = .0059). (C) Immunofluorescence analysis of EB1 and F-actin. 2D representation of intensity profile of EB1 and F-actin in the region of interest indicated by the box were analyzed in ImageJ Version 1.43J software. Scatter graph for ratio of intensity of EB-1 at back versus front (identified using F-actin). Thirty cells were analyzed from individual experiment and 3 independent experiments were performed (*P = .003). (D) WT neutrophils were transfected with GFP-EB1CΔAC or GFP plasmid vector control using nucleofactor and stimulated with fMLP and Fg and stained for F-actin. Histogram is cells with more than 1 protrusion. Fifty cells were analyzed from individual experiment and 3 independent experiments were performed (mean ± SD; *P = .0027). (E) Immunofluorescence analysis of F-actin and microtubules after stimulation. Scatter plot for MT polarity quantification (mean ± SD; **P = .000017). (F) Western blots of WCL probed with Glu-tubulin (**P = .0024). In fluorescence images tubulin and EB-1 were stained with secondary antibody conjugated with Alexa Fluor 488, while F-actin was labeled with Rhodamine Phalloidin and slides were mounted in SlowFade Gold Antifade. Fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63× objective N/A1.3 with ORCA-ER C4742-95 camera driven by Openlab Version 5.5.0 software.

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