Fig. S4.

Adjusting Ecad-Fc ligand density does not affect cell spreading, morphology, or E-cadherin organization on 60-kPa PA gels. (A) Representative images of 30-kPa and 60-kPa Ecad-Fc PA gels prepared with varying concentrations of SS stained with an anti–E-cadherin antibody. (Scale bar, 50 µm.) (B) Quantification of anti–E-cadherin antibody staining on the surface of functionalized gels. Data represent the fluorescence intensities normalized to the “30 kPa, 2.0 mg/mL” condition averaged from three independent experiments. Error bars represent SEM. Statistics were determined using a Student’s t test, *P < 0.05. (C) Representative images of E-cadherin:dsRed MDCK cells on 30-kPa and 60-kPa PA gels functionalized with varying concentrations of SS. Cells were fixed and stained with phalloidin. (Scale bar, 10 µm.) (D) Average cellular E-cadherin fluorescence intensity on 30-kPa and 60-kPa PA Ecad-Fc gels functionalized with varying concentrations of SS normalized to the 30 kPa, 2.0 mg/mL condition from three independent experiments. Error bars represent SEM. Statistics were determined using a Student’s t test. (E–G) Quantification of morphological features, including cell spread area (E), aspect ratio (F), and circularity (G). Cumulative data from three independent experiments are represented in a dot plot, where each dot represents one cell. The solid black line indicates the median and upper and lower bars represent the interquartile range (n > 80 cells per condition). Statistics were determined using a Kruskal–Wallis test with Dunn’s posttest for multiple comparisons, ****P < 0.0001. (H) Representative SIM superresolution images of cells stably expressing E-cadherin:dsRed and stained for actin (phalloidin) on 30-kPa and 60-kPa PA Ecad-Fc gels functionalized with varying concentrations of SS. (Scale bar, 10 µm.) (I) Quantification of E-cadherin puncta per square micrometer represented in a box and whisker format, in which the ends of the box mark the upper and lower quartiles, the horizontal line in the box represents the median, and whiskers outside the box extend to the highest and lowest value within the 1.5× interquartile range. Data shown are cumulative from three independent experiments. (J) Quantification of E-cadherin puncta size represented in a box and whisker format, in which the ends of the box mark the upper and lower quartiles, the horizontal line in the box represents the median, and whiskers outside the box extend to the highest and lowest value within the 1.5× interquartile range. Data shown are cumulative from three independent experiments. For quantification (I and J), five 1-µm² regions within cell protrusions were analyzed per cell and at least 10 cells per condition were quantified. Statistics were determined using a Kruskal–Wallis test with Dunn’s posttest for multiple comparisons.