*Cell Culture*

Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in Medium 200 supplemented with 1% penicillin-streptomycin (Corning) and Large Vessel Endothelial Supplement (LVES) solution. HUVECS, Medium 200, and LVES were purchased from ThermoFisher. HUVECs from passages 12–14 were cultured in 0.1% gelatin-coated flasks at 37o C and 5% CO2 prior to running all the experiments.

*Polyacrylamide Gel Fabrication*

The protocol for preparing Polyacrylamide (PA) gels can be found in [30]. In brief, glass bottom petri dishes (35 mm, Cellvis) were treated with bind silane solution for 45 minutes, rinsed with DI water, and air-dried. The PA solution is made by mixing ultra-pure water, 40% acrylamide (Bio-Rad), 2% bis-acrylamide (Bio-Rad), and fluorescent beads (Texas red with 0.5 µm diameter, Invitrogen). The PA solution was mixed and placed in a vacuum chamber for 40 minutes. After this time, polymerization was initiated by adding 10% ammonia persulfate and TEMED (N,N,N′,N′- tetramethylethane-1,2-diamine) to the PA gel solution, which was subsequently added to the petri dish. Finally, hydrophobic coverslips were placed on top of the PA solution to allow complete polymerization of the PA solution to a PA gel. Our gels had a stiffness of approximately 1.2 kPa and height of approximately 100 µm [31].

*Cellular Micropattern Preparation*

Polydimethylsiloxane (PDMS) was used to fabricate thin micropatterns as described previously in [30–32]. In brief, a thin cross-section of PDMS (Dow Corning) was prepared by mixing silicone base with a curing agent (20:1) and the mixture was then poured into a 100 mm petri dish. The PDMS mixture in the petri dish was then incubated at 70o C overnight. Thin, circular cross-sections of the cured PDMS (16 mm) were fabricated using a hole puncher. Small holes (5–6 micropatterns) were made on the circular PDMS section using a biopsy punch (world precision instruments) with diameter ~ 2 mm each.

*SANPAH Burning & ECM Surface Coating*

The petri dish samples with PDMS micropatterns stamped on PA gels were subject to treatment with sulfosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (Sulfo-SANPAH; Proteochem) dissolved in 0.1 M HEPES buffer solution (Fisher Scientific) and exposed to UV light for at least 8 mins. After the UV treatment, SANPAH was rinsed off the PA gel approximately 2–3 times using PBS and the PA gels were coated with ECM at a total concentration of 0.1 mg/mL at one of the following ratios and compositions; 1) 100% Col-I (0.1 mg/ml), 2) 75% Col-I and 25% FN, (0.075 mg/ml & 0.025 mg/ml), 3) 50% Col-I and 50% (0.05 mg/ml & 0.05 mg/ml) FN, 4) 25% Col-I and 75% FN (0.025 mg/ml & 0.075 mg/ml), and 5)100% FN (0.1 mg/ml). ECM-coated gels were placed in the refrigerator overnight at 4o C and after this time excess ECM protein solution was carefully removed and HUVECs were seeded at a density of ~ 50 × 104 cells/mL. After 60–75 mins, micropatterns were carefully removed from the PA gel leaving us with circular 2 mm diameter circular patterns. The circular HUVEC monolayers were incubated at 37o C and 5% CO2 for at least 24 hours prior to experimentation.

*Time Lapse Microscopy*

An inverted Zeiss microscope equipped with a 5x objective & Hamamatsu camera was used to acquire brightfield images and fluorescent images at 5-minute intervals for 3 hours.

*Traction Force Microscopy & Monolayer Stress Microscopy*

Traction force microscopy (TFM) and monolayer stress microscopy (MSM) were used to calculate tractions and intercellular stresses, respectively [8, 11, 12, 30]. Briefly, the cell-induced deformations on the top surface of the gel were calculated by running a custom-written, window-based particle image velocimetry routine that computes the pixel shift of fluorescent images of the gel (cells attached) with respect to the stress-free reference image (fluorescent image taken after cell trypsinization). These displacements were then used to calculate tractions as shown by Butler et al. [11]. A window size of 32 and overlap of 0.75 was used. Building upon TFM, MSM was used to calculate intercellular stresses as previously described by Trepat et al. [9]. The intercellular stresses were recovered from traction force maps on the substrate by using a straightforward force balance imposed by Newton's law. The computed local two-dimensional stress tensor within the monolayer was converted into maximum principal stress (σmax) and minimum principal stress (σmin) along the principal plane by rotating the local coordinate system along the principal orientation. The maximum principal stress and minimum principal stress was used to calculate the average normal stress (σmax + σmin)/2 and maximum shear stress (σmax - σmin)/2. A cropped section of 651 µm × 651 µm was used for the analysis of tractions and intercellular stresses.

*Measurement of Cell Velocity*

Cellular velocity of HUVECs in the monolayer was computed using a PIV routine custom written in MATLAB. Cell displacements were calculated from pixel shift between phase images at two consecutive time points. The velocity map of the cells in the monolayer was calculated by averaging the change in displacements over the time interval. Cell velocity was calculated at every 5 min interval for the entire sequences of images acquired over 3 hours. A cropped section of 651 µm × 651 µm was used for the analysis mentioned above.

*Cell Area and Circularity measurements*

Cell area and cell circularity were measured using a custom feature extraction function written in MATLAB. This algorithm, which utilizes the image processing toolbox allows us to calculate the properties mentioned above by first converting phase contrast images to binary images, which are then segmented so each individual cell can be identified. Second each cell is assigned a number, and lastly, we extract cell area and circularity, from our binary images using MATLAB. Our algorithm calculates the cell area as the number of pixels contained in a region (sq. pixels) and converts this to µm2 based on a pixel to micron conversion factor. The circularity, which is equal to (4 × π × area)/(perimeter2) is a dimensionless number that tells us how close the cell’s shape is to a circle. Circularity values range from 0 to 1 with a value of 1 representing a perfect circle, while values further from 1 represent a deviation from a circular shape.

*Statistical Analysis*

The number of cells analyzed were 1057 to 1460 cells for each ECM coating concentration ratio. Each Col- I and FN coating ratio (75% Col-I − 25%FN, 50% Col-I − 50% FN, 25% Col-I − 75% FN, and 100% FN) was tested for statistical significance with respect to the 100% Col-I condition using ANOVA: Single Factor hypothesis test for cell area, cell orientation, and circularity respectively. The p-values for significance were calculated with an alpha level of 0.05 (Null hypothesis rejected for p < 0.05).