# Title of Dataset:  Molecular-scale substrate anisotropy, crowding, and division drive collective behaviors in cell monolayers

This dataset contains the data presented in the main text figures and supplemental information for “Molecular-scale substrate anisotropy, crowding, and division drive collective behaviors in cell monolayers”, by authors Yimin Luo, Mengyang Gu, Minwook Park, Xinyi Fang, Younghoon Kwon, Juan Manuel Uruenã, Javier Read de Alaniz, Matthew E. Helgeson, M. Cristina Marchetti and Megan T. Valentine published in the Journal of the Royal Society Interface.

## Description of the data and file structure

Data are all provided in comma separated value form (CSV). File names refer to the figures numbers of the manuscript.  If the letter S is provided before the number, the figure can be found in the Supplemental Materials. 

Main text

Figure 1. Schematic of the experimental approach. No quantitative data are presented.

Figure 2. Snapshots of cell configurations and associated angle distributions.
2a-c contain image data, no quantitative data are presented.
2e-f data format is specified below:
	FRAME: The frame sampled from the dataset
	XM: Fit ellipse coordinate x
	YM: Fit ellipse coordinate y
	Major: Fit ellipse major axis
	Minor: Fit ellipse minor axis
	Angle: Angle of the ellipse in radian, with range [0, π]

Figure 3. Evolution of the cell-substrate order parameter Scs with cell density ρ. Columns represent:
	“substrate type” – isotropic or nematic
	“day after seeding” – represented by different colors
	“S_cs” – Cell-substrate order parameter, labeled with respect to the substrate orientation: Scs = < 2 cos^2 θ_i – 1>
	 “S_cs_err” – standard error of the cell-substrate parameter

Figure 4. Analysis of spatial correlations of cell-cell orientation: 
4a,c data format is specified below:
	First row represents the density.
	First column represents the radius.
	Each column represents correlation for a given density.
	Each row represents correlation for a given radius.

These data are fit to either:
	an exponential expression C_θθ(r) = A_e exp(-r/ξ_θθ ) + C_∞ 
	a power law expression C_θθ(r) = A_p r^{−γ}

The fitting parameter and their upper- and lower-bounds (standard error intervals, shaded regions) are plotted in 4bcd:

4b data format is specified below:
Columns represent ρ, ξ_θθ, ξ_θθ_lowerbound, ξ_θθ_upperbound, substrate type

4d data format is specified below:
Columns represent ρ, γ, γ_lowerbound, γ_upperbound

4e data format is specified below:
Columns represent ρ, C_∞, C_∞_lowerbound, C_∞_upperbound

Figure 5. Spatial maps of the cell velocity field, color-coded by the instantaneous direction of each velocity vector.	
5ab data format is specified below:
	vx: instantaneous displace (between 2 frames) in x direction
	vy: instantaneous displace (between 2 frames) in y direction
	px: position
	py: position
5cd data format is specified below:
Velocity angle: velocity angle formed by atan2(vy/vx), with range [-π, π]

Figure 6. Angular distribution of the cell division axis. 
6a contains image data, no quantitative data are presented. 
6b-d data format is specified below:
Angle: cell division orientation angle in radian, with range [0, π]

Figure 7. Movement of defect as denoted by local director field.
a-d shows local director fields which is overlaid on the cytoplasm channel, data format is specified below:
	FRAME: the frame sampled from the dataset
	XM: Fit ellipse coordinate x
	YM: Fit ellipse coordinate y
	Major: Fit ellipse major axis
	Minor: Fit ellipse minor axis
	Angle: Angle of the ellipse in radian, with range [0, π]
7ef contain image data, no quantitative data presented.

Supplementary Materials

Figure S1, S2, S4, S15, S16, S17: no quantitative data are presented. For detailed information, please refer to the Supplementary Materials associated with the main text.

Figure S3. Angles of the nuclei are highly aligned with the cell body.
S3a contain image data, no quantitative data presented.
S3b shows scattering plot (in degrees) tallied from well-separated cells showing both angles are highly correlated or off by 180, data format is specified below:
	Each row represents an individual cell.
	Nucleus orientation:  Angle of the nucleus major axis with respect to x-axis, which in the case of nematic substrates represents alignment to the nematic director. Units are degrees.
	Body orientation: Angle of the cell major axis with respect to x-axis, which in the case of nematic substrates represents alignment to the nematic director. Units are degrees.

Figure S5. Cell-cell order parameter in relation to aspect ratio and cell-substrate order parameter
	S5a aspect ratio versus cell-substrate order parameter: Scc =< 2 cos^2 θ_ij – 1>. Data format is specified below: First column denotes S_sc, second and third column denote the average and standard deviation of the aspect ratio.
	S5bc cell-cell order parameter S_cc vs density or cell-substrate order parameter, data format is specified below: csv file contains data with labeled title, “density”, “S_cc”, “S_cs”, “substrate type”, “days after seeding”. Note: “days after seeding” is represented by different color, same as main text. 

Figure S6. additional fitting parameters, using approaches and metrics described in Figure 4.

Figure S7. Velocity correlations and anisotropy. vx, vy, and vx/vy. vmean, vxmean, vymean denote the ensemble average for all cells in a frame at time T.
	S7a Columns represent “distance r”, and pair correlation functions: “Cvv (nematic)” and “Cvv (isotropic)”
	S7b Columns represent a value of the acos(beta_ij), where beta_ij is difference in angle between all pairs. 
	S7e Columns represent “density”, “vx/vy”, “substrate type” and “day after” (labeled in different colors)
	S7f and S7g Columns represent “Time”, “vmean”, “vxmean”, “vymean”

Figure S8. Velocity Autocorrelation Functions (VAF) as a function of lag time and the mean velocity |V| at different cell densities.
	S8a Columns represent “lag time”, “VAF-nematic high density”, “VAF-nematic low density”, “VAF-isotropic high density”
	S8b: Columns represent “avg density”, “density deviation”, “mean velocity”, “velocity deviation”, “substrate type”, “density”

Figure S9. Effects of the box size on the measurement of cell-substrate correlations.
	S9a is a schematic and contains no quantitative data.
	S9b,c: Columns represent the average number of cells in the a box of size LxL “<N>”, and its standard deviation “∆N”, the averaged cell-substrate order parameter “<S_cs>”, and its standard deviation “∆S_cs”, for three snapshots at different conditions, purple: nematic (N = 4200), orange: nematic (N = 2200) and yellow: isotropic (N = 2200). For this calculation, L = [500, 1000, 1500, 2000, 2500, 3000] μm.


Figure S10. Giant number fluctuations in the cellular density, color-coded by density. S10ab Columns represent “N_mean” – average number for a given box size, “N_std”- standard deivation for particles in a box of given box size, then we compute “N_std./sqrt(N_mean)” and “density” for S10a (isotropic), S10b (nematic).

Figure S11. Differential scanning calorimetry to determine crystallization peak and iso-nematic
transition temperature of the mixture. csv contains temperature history (both up and down) and exothermic, endothermic heat flow. Columns represent “T(up)”, “Q(up)”, “T(down)”, “Q(down)”.

Figure S12. Wide angle X-ray scattering data to find the order parameter of the aligned substrate. Columns represent “angle” and “intensity” for isotropic and nematic substrates.

Figure S13. Dynamic mechanical analysis performed on the nematic and isotropic substrates.
Here we include the time sweep data output from the dynamic mechanical analysis, as described in the SI.  The mean and standard deviation of each dataset are plotted in Figure S12.

Figure S14. Surface roughness characterization, both AFM and SEM data are presented here.
S14a: AFM Columns A and B represent the x- and y-positions of the AFM tip during scanning and Column C represents the height (z-position). 
S14b contains no quantitative data.

Figure S18. Analyzing the orientation of fibronectin deposition for isotropic (a)(c) and nematic
(b)(d) substrates. (a-b) contain no quantitative data.
S18c, S18d data format is specified below (analyzed by Image J):
First column denotes the average angle of the binned directional count (“Direction”), second column denotes the density of that value (“Amount”), third column denotes the smooth fit of “Amount”, which is shown as a solid line on the plot. 

Figure S19. Comparison of density, order parameter and velocities for cells treated with focal adhesion kinase inhibitor to those that have not been treated. Data format is specified below:
	S19ae contain no quantitative data.
	S19bc (FAKi-treated case): columns represent “time”, “density”, “cell-substrate order Scs”
	S19d (FAKi-treated case): columns represent “time”, “vmean”, “vxmean”, “vymean”
	S19fg (no FAKi control): columns represent “time”, “density”, “cell-substrate order Scs”
	S19h (no FAKi control): data replotted from Figure S7f







