Figure 5.
Figure 5. Arf6-dependent integrin recycling controls TF signaling and procoagulant function. HaCaT cells were pre-incubated with small-molecule inhibitors QS11 and SH3 for 2 hours. QS11 promotes arf6-GTP loading and thus it promotes arf6 activity, whereas SH3 inhibits arf6-GEF cytohesin, which causes accumulation of nonactive arf6-guanosine diphosphate. (A) Confocal imaging of HaCaT cells treated with QS11 (10 μM) or SH3 (20 μM). FVIIa (10 nM) was added together with FVIIa antibody 12C7-Alexa647 (red), and cells were incubated for 5 or 30 minutes. Upon fixation, cells were counterstained for early endosome antigen 1 (EEA1-Alexa488, green) and nuclei (Hoechst, blue). Scale bar = 10 μm. (B) TS2/16 immunoprecipitates and debris-cleared lysates (CLs) of Brij 35 cell lysates of HaCaT cells treated with QS11 (10 μM) and SH3 (20 μM) before FVIIa stimulation (10 nM; 30 minutes) were analyzed by western blot for TF and integrin β1. (C) FXa generation on HaCaT cells treated with QS11 and SH3 for 2 hours (n = 4). *P < .05, **P < .01, one-way ANOVA and Dunnett’s test. (D) Representative surface staining for FVIIa after fixation with antibody 3G12 following exposure of MDA-MB-231 to FVIIa with or without SH3 pretreatment. (E) Quantification of fluorescent intensity of at least 4 random views from 2 independent experiments of cells treated as shown in panel D. Fluorescent intensity of images was quantified with imageJ software. (F) FXa generation on MDA-MB-231 cells as described in panel C. *P < .05. (G) ERK phosphorylation of HaCaT cells pretreated with arf6 inhibitors before stimulation with SLIGRL (50 μM) or FVIIa (10 nM) for 30 minutes. (H) Quantification of IL-8 mRNA induction in QS11- and SH3-pretreated HaCaT cells after 90 minutes of stimulation with SLIGRL (50 μM) or FVIIa (10 nM) (n = 4). ***P < .001, one-way ANOVA and Tukey’s test. (I) Representative confocal images of HaCaT cells stimulated with FVIIa (10 nM) for 30 minutes, fixed, permeabilized, and stained for TF (5G9-Alexa647) or integrin β1 (TS2/16-Alexa647). Scale bar = 10 μm. (J) FACS surface staining of TF and integrin β1 on HaCaT cells that were stimulated with FVIIa for 30 minutes (n = 7).

Arf6-dependent integrin recycling controls TF signaling and procoagulant function. HaCaT cells were pre-incubated with small-molecule inhibitors QS11 and SH3 for 2 hours. QS11 promotes arf6-GTP loading and thus it promotes arf6 activity, whereas SH3 inhibits arf6-GEF cytohesin, which causes accumulation of nonactive arf6-guanosine diphosphate. (A) Confocal imaging of HaCaT cells treated with QS11 (10 μM) or SH3 (20 μM). FVIIa (10 nM) was added together with FVIIa antibody 12C7-Alexa647 (red), and cells were incubated for 5 or 30 minutes. Upon fixation, cells were counterstained for early endosome antigen 1 (EEA1-Alexa488, green) and nuclei (Hoechst, blue). Scale bar = 10 μm. (B) TS2/16 immunoprecipitates and debris-cleared lysates (CLs) of Brij 35 cell lysates of HaCaT cells treated with QS11 (10 μM) and SH3 (20 μM) before FVIIa stimulation (10 nM; 30 minutes) were analyzed by western blot for TF and integrin β1. (C) FXa generation on HaCaT cells treated with QS11 and SH3 for 2 hours (n = 4). *P < .05, **P < .01, one-way ANOVA and Dunnett’s test. (D) Representative surface staining for FVIIa after fixation with antibody 3G12 following exposure of MDA-MB-231 to FVIIa with or without SH3 pretreatment. (E) Quantification of fluorescent intensity of at least 4 random views from 2 independent experiments of cells treated as shown in panel D. Fluorescent intensity of images was quantified with imageJ software. (F) FXa generation on MDA-MB-231 cells as described in panel C. *P < .05. (G) ERK phosphorylation of HaCaT cells pretreated with arf6 inhibitors before stimulation with SLIGRL (50 μM) or FVIIa (10 nM) for 30 minutes. (H) Quantification of IL-8 mRNA induction in QS11- and SH3-pretreated HaCaT cells after 90 minutes of stimulation with SLIGRL (50 μM) or FVIIa (10 nM) (n = 4). ***P < .001, one-way ANOVA and Tukey’s test. (I) Representative confocal images of HaCaT cells stimulated with FVIIa (10 nM) for 30 minutes, fixed, permeabilized, and stained for TF (5G9-Alexa647) or integrin β1 (TS2/16-Alexa647). Scale bar = 10 μm. (J) FACS surface staining of TF and integrin β1 on HaCaT cells that were stimulated with FVIIa for 30 minutes (n = 7).

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