Volume 110, Issue 8, Pages 1845-1857 (April 2016) Active Traction Force Response to Long-Term Cyclic Stretch Is Dependent on Cell Pre- stress  Heather Cirka, Melissa Monterosso, Nicole Diamantides, John Favreau, Qi Wen, Kristen Billiar  Biophysical Journal  Volume 110, Issue 8, Pages 1845-1857 (April 2016) DOI: 10.1016/j.bpj.2016.02.036 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Overview of the method. (a) Wells were created in a custom polycarbonate mold out of 16:1 PDMS. (b) PA gels were attached to PDMS wells according to a previously published protocol. After chemical treatment that activated the silicone substrate, an unpolymerized droplet of PA was placed onto a well. A 22 mm coverslip was placed onto a droplet (left) and a uniform gel thickness was acquired by capillary action. After polymerization, the coverslip was removed (right). (c) Stretch wave form of the strain-field analysis. Colored dots indicate where representative images of the wells were taken, and they are outlined for clarity. (d) The analysis region of the uniaxially stretched gel for strain-field analysis is indicated by the transparent red square. Longitudinal (εxx) and lateral (εyy) strain fields are shown. To see this figure in color, go online. Biophysical Journal 2016 110, 1845-1857DOI: (10.1016/j.bpj.2016.02.036) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Overview of traction force measurements. (a) Time course schematic of experiments. All stretch experiments were at 10% magnitude (either uniaxial or biaxial stretch). At 24 h, stretch was stopped and the well was removed from the stretch device and placed in the microscope viewing dish for image acquisition. Wells were in their original conformation during the image acquisition process. (b) Side view of the image acquisition setup. (c) Representative images of traction force calculation. Phase images of the cell were acquired, as were fluorescent images of beads embedded within the PA gel. Green and red colors show the stressed and relaxed (cells trypsinized) substrate, respectively. Using a particle-tracking algorithm, bead displacements were calculated. Finally, knowing the modulus of the substrate and the bead displacements, a stress map of the substrate was created. To see this figure in color, go online. Biophysical Journal 2016 110, 1845-1857DOI: (10.1016/j.bpj.2016.02.036) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Overview of experiments. VICs were cultured on 7.5 kPa substrates unless otherwise indicated. Pretreatments are indicated on the representative static stress maps. Arrows indicate whether uniaxial (straight arrows) or biaxial stretch (bisecting arrows) was used for a given experiment. For cells cultured under high pre-stress conditions, mean traction force, mean contractile moment, mean cell area, and mean maximal substrate stress all decreased with stretch. The mean form factor increased for both equibiaxially stretched cells and TGF-β1-pretreated cells when uniaxially stretched indicating a decrease in extent of cell extensions. The opposite was true for cells under low pre-stress conditions: mean traction force, mean maximal substrate stress, and mean cell area all increased when cells were stretched compared to static control cells. Scale bar, 50 μm. To see this figure in color, go online. Biophysical Journal 2016 110, 1845-1857DOI: (10.1016/j.bpj.2016.02.036) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 Cell traction force and area change with long-term cyclic stretch. (a) The percentage change in cell traction force was normalized to respective controls for each treatment group. Error bars reflect raw stretch traction force values divided by the mean of the controls then multiplied by 100%. Cell traction force decreases with biaxial stretch, uniaxial stretch, and uniaxial stretch with TGF-β1 pretreatment (TGFβ) on a 7.5 kPa substrate. Cell traction force increases with stretch slightly when cells are pretreated with 10 μM blebbistatin (Blebb) before stretch and with cells that are stretched on a soft substrate. (b) The percentage change in cell area was normalized to respective controls for each treatment group. Error bars reflect the raw stretch cell area values divided by the mean of the controls. Cell area decreases with biaxial stretch, uniaxial stretch, and uniaxial stretch with TGF-β1 pretreatment on a 7.5 kPa substrate. Cell area increases with stretch when cells are pretreated with 10 μM blebbistatin and subsequently stretched and with cells that are stretched on a soft substrate. (c) The percentage change in contractile moment followed the same trends as that of traction force, with the average contractile moment decreasing significantly for cells under high pre-stress. For cells under low pre-stress, there was an increase in the average contractile moment with stretch. The asterisk indicates significance when compared via Student’s t-test to respective static controls (p < 0.05). To see this figure in color, go online. Biophysical Journal 2016 110, 1845-1857DOI: (10.1016/j.bpj.2016.02.036) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 Changes in cell shape and elongation with long-term cyclic stretch. (a) Elongation ratio was unaffected by stretch under most treatment conditions, except in the case of culture on a soft (0.6 kPa) gel. (b) Form factor increased with biaxial stretch and uniaxial stretch for TGF-β1-pretreated cells (TGFβ) as the cell perimeter decreased with depolymerization of many cellular extensions in the direction of stretch. Form factor decreased with stretch on a soft substrate as cells elongated from a circular morphology. ∗p < 0.05. To see this figure in color, go online. Biophysical Journal 2016 110, 1845-1857DOI: (10.1016/j.bpj.2016.02.036) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 6 Angle of orientation for static and stretched cells represented as 0–90° histograms. The angle of orientation was measured with respect to the stretch direction (0–180°). For cells that had an orientation angle above 90°, the supplementary angle was reported. Cells that oriented perpendicular to the stretch had an angle of orientation of 90°. (a) biaxially stretched cells compared to static controls (b) uniaxially stretched cells compared to static controls (c) TGF-β1 pre-treated cells stretched uniaxially compared to TGF-β1 pre-treated static controls (d) Blebbistatin pre-treated cells stretched uniaxially compared to Blebbistatin static controls (e) Cells cultured on 0.6 kPa substrates stretched uniaxially compared to static control cells cultured on 0.6 kPa. For all panels, black bars are static cells, gray bars are stretched cells. Biophysical Journal 2016 110, 1845-1857DOI: (10.1016/j.bpj.2016.02.036) Copyright © 2016 Biophysical Society Terms and Conditions