Data rom: Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment

The ability of cells to organize into tissues with proper structure and function requires the effective coordination of proliferation, migration, polarization, and differentiation across length scales. Skeletal muscle is innately anisotropic; however, few biomaterials can emulate mechanical anisotropy to determine its influence on tissue patterning without introducing confounding topography. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively polarize in the stiffest direction. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanica..., , , # Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment

[https://doi.org/10.5061/dryad.08kprr59c](https://doi.org/10.5061/dryad.08kprr59c)

Dataset contains replicate data for all figures presented in the main text and supplemental materials.

## Description of the data and file structure

Each figure has an associated .xlsx file with all replicate data and annotations if applicable.

Fig. 1B: Elastic modulus parallel and orthogonal to mLCN nematic director derived from the initial linear regime of stress-strain curves. Stiffness anisotropy ratio and difference is calculated from the mean of repeated tensile tests.

Fig. 1E/F: Myotube orientation-order parameter (*S*) and nematic correlation length (µm) of myotubes after 5 days of differentiation on isotropic and aligned substrates.

Fig. 2B: Myoblast migration speed (µm/hr) and normalized migration ± 10˚ from nematic director on mLCNs through confluence.

Fig. 2C/D: (C) Temporal evolutio...