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. Author manuscript; available in PMC: 2018 Nov 29.
Published in final edited form as: Converg Sci Phys Oncol. 2017 Nov 29;3:044001. doi: 10.1088/2057-1739/aa9263

Figure 3.

Figure 3

Matrix stiffness and VEGF stimulation foster VEGFR-2 colocalization with EEA-1 and decrease cell surface VEGFR-2 levels in endothelial cells. (A) Sub-confluent HUVECs on 1 kPa, 10 kPa, or 10 kPa collagen-coated gels with Y-27632 pretreatment were cultured under control conditions (left panels) or stimulated with 15 min VEGF (right panels) and then co-stained for VEGFR-2 and EEA-1. Insert is a 3x zoom of the boxed region. Images were acquired with identical exposure settings (Scale bars, 20 μm). (B) The average number of VEGFR-2 and EEA-1 colocalization events per cell compared to control conditions. Plots are mean ± SE. N = 3 independent experiments, at least 30 cells per condition. (C) Non-permeabilized HUVECs were stained for VEGFR-2 following exposure to control conditions (left panel) or 15 min VEGF treatment (right panel) to visualize receptors located on the cell surface. Insert is a 3x zoom of the boxed region. Arrows indicate VEGFR-2 clusters formed after VEGF stimulation. Images were captured with identical exposure settings (Scale bars, 20 μm). (D) Quantification of the average number of membrane-bound VEGFR-2 clusters per condition. Plots are mean ± SE. N = 3 independent experiments, at least 30 cells per condition. * p<0.05, ** p<0.01, *** p<0.001 from ANOVA with post hoc Tukey’s test. N.S. not significant.