Cell spreading to collagen I. Transduced cells were incubated on collagen I coated glass cover slips, fixed and treated with the F-actin binding protein phalloidin. Images were retrieved using a fluorescence Nikon eclipse TE300 inverted microscope with a Plan Fluor ELWD 40x/0.60 NA objective (Nikon Tokyo, Japan) attached to a Nikon DS-5M-L1 Camera system. The cell spreading was quantified by measuring the area of individual cells in a randomly picked field using ImageJ. Results are presented as mean cell area + 95% CI of one representative experiment of two to three. (A) MDA-MB-231 cells (n ≥ 19, **p = .0017 and ***p < .0001 against pSiRPG) and (B) Sum102 cells (n ≥ 5, *p = .0137 and ***p < .0001 against pSiRPG) with TFPI downregulated. (C) MDA-MB-231 with TFPIβ downregulated (n ≥ 19, ***p < .0001 against pSiRPG). (D) Visualization of the actin stress fibers in MDA-MB-231 (upper panels) and Sum102 (lower panels) cells expressing shRNA4 (left), -6 (middle left), and -7 (middle right) against TFPI or pSiRPG control vector (right) and in (E) MDA-MB-231 cells expressing shRNA7β (left) and -9β (middle) against TFPIβ or empty control vector (right). Scale bar = 20 μm.