NMIIA is essential for traction force generation during initial adhesion and spreading. (A) NMIIA and NMIIB protein expression levels in 4T1 cells stably infected with NT lentiviral constructs or shRNA constructs to deplete NMIIA or NMIIB. (B) Diagram demonstrating TFM. Fluorescent microbeads are embedded within a PAA gel formed on a glass coverslip. The PAA gel is then coated in a continuous layer of 50 µg/ml fibronectin. Plated cells deform the gel during spreading, moving the embedded beads. The cells are then trypsinized, allowing for the relaxation of the gel and return of the beads to their original position. The distance of bead movement is measured to quantify traction stress. (C) 4T1 cell traction force generation during active spreading. Traction stress was measured every 15 min for 1 h after plating. Representative images of control, NMIIA-shRNA, and NMIIB-shRNA cells during the spreading time course are shown. Representative traction stress heat maps (top) and their corresponding phase-contrast image (bottom) are shown. (D) Traction stresses are quantified by averaging the magnitude of all calculated vectors within the area of the cell outline. The mean of the per cell average traction stress was quantified. Error bars are SEM; n = 20 cells.