Figure 6
Figure 6. EphA2 colocalizes with ICAM-1 and VCAM-1 after in vitro EFNA4-Fc treatment of HUVECs. (A) HUVEC monolayers grown onto glass chamber slides and overnight activated with TNF-α (10 ng/mL) were incubated for 60 minutes with Alexa Fluor 405–EFNA4-Fc protein complexes. After cell fixation of cultures (at 15, 30, or 60 minutes), confocal images were acquired from the apical to the basal side of cells (0.1-μm z-steps; 63× immersion-oil objective). Pseudocolor Z-series projections of image stacks were created (Leica LCS software) to determine the topologic distribution of EFNA4-Fc spots at each time point (left). At each time point, the number of basal and apical EFNA4-Fc spots was determined through image segmentation and analysis (MetaMorph version 7.1; right), according to the color threshold established in the pseudocolor scale bar (red regions, apical; blue regions, basal). Images are from a representative experiment after 60 minutes of culture. (B) HUVECs monolayers grown as in panel A were treated with either EFNA4–Fc protein complexes (preclustered with biotinylated anti-His Ab followed by streptavidin–Alexa Fluor 405) or cross-linking anti–ICAM-1 mAb (anti–ICAM-1 primary Ab preclustered with anti–mouse Alexa Fluor 488 Ab, green). Cultures were fixed after 15, 30, or 60 minutes and immunostained to analyze the colocalization of clustered EphA2 (anti-EphA2 plus Alexa Fluor 546 secondary Ab) with ICAM-1 (Alexa Fluor 488 Ab) or VCAM-1 (Alexa Fluor 633 Ab) or of EphA2 (Alexa Fluor 546) or VCAM-1 (Alexa Fluor 633 Ab) with ICAM-1, respectively. Z-series confocal images were acquired as in panel A. Fluorescent images are from a representative experiment of EFNA4–Fc treatment. Fluorescent blue-red spots, corresponding to EFNA4-Fc aggregated EphA2 (left), were identified as objects through color thresholding (Metamorph software; white regions). The percentage of these regions containing ICAM-1 (green, right), was measured to determine EphA2 colocalization. The graphs represent the results from the corresponding treatments.

EphA2 colocalizes with ICAM-1 and VCAM-1 after in vitro EFNA4-Fc treatment of HUVECs. (A) HUVEC monolayers grown onto glass chamber slides and overnight activated with TNF-α (10 ng/mL) were incubated for 60 minutes with Alexa Fluor 405–EFNA4-Fc protein complexes. After cell fixation of cultures (at 15, 30, or 60 minutes), confocal images were acquired from the apical to the basal side of cells (0.1-μm z-steps; 63× immersion-oil objective). Pseudocolor Z-series projections of image stacks were created (Leica LCS software) to determine the topologic distribution of EFNA4-Fc spots at each time point (left). At each time point, the number of basal and apical EFNA4-Fc spots was determined through image segmentation and analysis (MetaMorph version 7.1; right), according to the color threshold established in the pseudocolor scale bar (red regions, apical; blue regions, basal). Images are from a representative experiment after 60 minutes of culture. (B) HUVECs monolayers grown as in panel A were treated with either EFNA4–Fc protein complexes (preclustered with biotinylated anti-His Ab followed by streptavidin–Alexa Fluor 405) or cross-linking anti–ICAM-1 mAb (anti–ICAM-1 primary Ab preclustered with anti–mouse Alexa Fluor 488 Ab, green). Cultures were fixed after 15, 30, or 60 minutes and immunostained to analyze the colocalization of clustered EphA2 (anti-EphA2 plus Alexa Fluor 546 secondary Ab) with ICAM-1 (Alexa Fluor 488 Ab) or VCAM-1 (Alexa Fluor 633 Ab) or of EphA2 (Alexa Fluor 546) or VCAM-1 (Alexa Fluor 633 Ab) with ICAM-1, respectively. Z-series confocal images were acquired as in panel A. Fluorescent images are from a representative experiment of EFNA4–Fc treatment. Fluorescent blue-red spots, corresponding to EFNA4-Fc aggregated EphA2 (left), were identified as objects through color thresholding (Metamorph software; white regions). The percentage of these regions containing ICAM-1 (green, right), was measured to determine EphA2 colocalization. The graphs represent the results from the corresponding treatments.

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