1. Cell-Cell Adhesion and Cortical Actin Bending Govern Cell Elongation on Negatively Curved Substrates 
Ai Kia Yip, Pei Huang, Keng-Hwee Chiam 
Biophysical Journal 
Volume 114, Issue 7, Pages (April 2018)
DOI: /j.bpj
Copyright © 2018 Biophysical Society Terms and Conditions

2. Figure 1
(A) Schematic representation of setup to fabricate semicylindrical substrates. (B) Schematic representation of experimental protocol to obtain substrates with semicylindrical grooves of varying curvatures is shown. (C) Schematic representation of process to visualize the curvature of the resultant plastic mold is shown. (D) xy-, xz-, and yz-cross-sectional views of a radius of curvature Rc = 50 μm plastic mold mounted on top of a drop of fluorescein are shown. Scale bar represents 100 μm. (E) Schematic representation of the model cell with height h, length L, and projected angle θ adhering to an underlying curved substrate is shown. The hexagonal ring represents the cortical actin modeled. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2018 Biophysical Society Terms and Conditions

3. Figure 2
(A–C) Maximal projection of image z-stack of NIH3T3 fibroblasts on (A) a flat substrate, a cylinder substrate of radius of curvature Rc, (B) 40 μm, and (C) 20 μm, with corresponding xz-cross-sectional view below each image. The color blue corresponds to nucleus, green corresponds to E-cadherin, and red corresponds to F-actin. Scale bar represents 10 μm. (D–F) Maximal projection of image z-stack of MDCK cells on (D) a flat substrate, a cylinder substrate of radius of curvature Rc, (E) 40 μm, and (F) 20 μm, with corresponding xz-cross-sectional view below each image. Scale bar represents 20 μm. (G and H) Angular distribution of (G) NIH3T3 fibroblasts or (H) MDCK cells, on substrates of various radius of curvature Rc, with 0° and 90° being perpendicular and parallel to the long axis of the cylinder is shown. (I) Elongation of NIH3T3 and MDCK cells versus. radius of curvature Rc. ∗∗∗p < NIH3T3: n = 64, 44, 52, 104, 76 cells for Rc = 20, 40, 50, 100 μm, and flat substrates, respectively. MDCK: n = 184, 378, 587, 09, 2047 cells for Rc = 20, 40, 50, 100 μm, and flat substrates, respectively. Error bars indicate SEM. (J) Percentage of NIH3T3 and MDCK cells that lie within 20° of the long axis of the channel versus radius of curvature Rc is shown. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2018 Biophysical Society Terms and Conditions

4. Figure 3
(A–C) Maximal projection of image z-stack of MDCK cells treated with blebbistatin on (A) a flat substrate, a cylinder substrate of radius of curvature Rc, (B) 40 μm, and (C) 20 μm, with corresponding xz-cross-sectional view below each image. Blue corresponds to nucleus, green corresponds to E-cadherin, and red corresponds to F-actin. Scale bar represents 20 μm. (D) Angular distribution of MDCK cells treated with blebbistatin on substrates of various radius of curvature Rc, with 0° and 90° being perpendicular and parallel to the long axis of the cylinder, is shown. (E–G) Maximal projection of image z-stack of MDCK cells treated with calyculin A on (E) a flat substrate, a cylinder substrate of radius of curvature Rc, (F) 40 μm, and (G) 20 μm, with corresponding xz-cross-sectional view below each image, is shown. Scale bar represents 20 μm. (H) Angular distribution of MDCK cells treated with calyculin A, on substrates of various radius of curvature Rc, with 0° and 90° being perpendicular and parallel to the long axis of the cylinder, is shown. (I–K) Maximal projection of image z-stack of MDCK cells treated with EGTA on (I) a flat substrate, a cylinder substrate of radius of curvature Rc, (J) 40 μm, and (K) 20 μm, with corresponding xz-cross-sectional view below each image, is shown. Scale bar represents 20 μm. (L) Angular distribution of MDCK cells treated with EGTA, on substrates of various radius of curvature Rc, with 0° and 90° being perpendicular and parallel to the long axis of the cylinder, is shown. (M) Percentage of MDCK cells with various treatment that lie within 20° of the axis of the channel versus radius of curvature Rc is shown. Error bars indicate standard error of the mean. (N) Elongation of MDCK cells with various treatment versus radius of curvature Rc is shown. ∗ and ∗∗∗ represent p < 0.05 and p < 0.001, respectively. Control: n = 184, 378, 587, 509, 2047 cells for Rc = 20, 40, 50, 100 μm, and flat substrates, respectively. Blebbistatin: n = 916, 238, 612, 1879, 1253 cells for Rc = 20, 40, 50, 100 μm, and flat substrates, respectively. EGTA: n = 310, 176, 655, 350, 1072 cells for Rc = 20, 40, 50, 100 μm, and flat substrates, respectively. Calyculin A: n = 210, 136, 273, 335, 338 cells for Rc = 20, 40, 50, 100 μm, and flat substrates, respectively. Error bars indicate SEM. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2018 Biophysical Society Terms and Conditions

5. Figure 4
(A) Heat map showing simulation results obtained by varying the bending modulus and cell-cell adhesion strength. The different colored regions indicate different realms of stability: gray indicates elongation decrease with decreasing Rc, black indicates elongation increase with decreasing Rc, and dark gray indicates biphasic with peak elongation at Rc = 30–50 μm. (B) Model and experimental comparison of elongations of untreated MDCK cells with Rc (α′ = 700 μm2 and k′ = 4,300,000 μm5) is shown. (C) Model and experimental comparison of elongations of MDCK cells with lower cell-cell adhesion, where α′ = 380 μm2 and k′ = 4,300,000 μm5 (model) or treated with EGTA (experiment), is shown. (D) Model and experimental comparison of elongations of NIH3T3 fibroblasts with Rc (α′ = 0 μm2 and k′ = 5,000,000 μm5) is shown. The elongations in (B) and (D) were normalized by the cell elongation on the flat substrate. For the model results, the case where Rc = 500 μm was used as an estimate for flat substrates. Error bars indicate standard error of the mean. Overall, simulation data show strong agreement with experimental data. (E and F) Simulated distribution of cell angular alignment with respect to the long axis of cylinder for cells on substrates with different Rc for (E) MDCK cells and (F) NIH3T3 fibroblasts is shown. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2018 Biophysical Society Terms and Conditions