FIGURE 3 from Antibody–Drug Conjugate αEGFR-E-P125A Reduces Triple-negative Breast Cancer Vasculogenic Mimicry, Motility, and Metastasis through Inhibition of EGFR, Integrin, and FAK/STAT3 Signaling

αEGFR-E-P125A inhibits EGFR, FAK, and STAT3 phosphorylation. A, Phospho-antibody array depicts changes in phosphorylation from MDA-MB-231-4175 cells plated on matrigel and undergoing the tube formation process to those plated on matrigel and treated with αEGFR-E-P125A for 16 hours in which tube formation was inhibited (left). Zoomed panel depicts decreased EGFR Y1069, FAK Y397, and STAT3 Y705 phosphorylation upon αEGFR-E-P125A treatment (right). B, Bubble plot depicts top downregulated phospho-sites as seen in the phospho-antibody array. Downregulated phospho-sites of interest which correlate to transcriptomic findings include EGFR Y1069, FAK Y397, and STAT3 Y705 sites. C–E, Quantification of EGFR Y1069, FAK Y397, and STAT3 Y705 downregulation from phospho-array. F–H, Western blots demonstrating downregulation of EGFR Y1069, FAK Y397, and STAT3 Y705 upon αEGFR-E-P125A treatment compared with controls. I–K, Immunofluorescence staining at 20x magnification demonstrating inhibition of tube formation and downregulation of EGFR Y1069, FAK Y397, and STAT3 Y705 upon αEGFR-E-P125A treatment. Staining was quantified by percent fluorescent area. **, P < 0.01; ***, P < 0.001.