EWI-2 effects on α4β1-mediated cell spreading. (A) MOLT-4 cells stably transduced with either EWI-2–flag construct or vector control were plated on VCAM-Ig–coated coverslips (2.5 μg/mL). Original magnification, × 20. (B) Quantitation of results from panel A. For the supplemental time-lapse video, micrographs are taken at 30-second intervals for 65 to 85 minutes. (C) MOLT-4 spreading at different VCAM-Ig coating densities. Original magnification, × 20. (D) Quantitation of results from panel C. Percent spreading for EWI-2-transfected cells (black bars) and control cells (gray bars) is shown at different VCAM-1 coating densities. All results were reproduced in multiple independent experiments. Surface expression levels (in mean fluorescent intensity [MFI] units [control cells, EWI-2 transfectants]) were determined by flow cytometry for α4 integrin (180, 183); β1 integrin (206, 208); and CD81 (215, 210). mAbs were A4-PUJ1, TS2/16, and JS64, respectively.