Extended Data Fig. 6: Cell clusters do not deposit additional ECM during migration.
From: Self-generated gradients steer collective migration on viscoelastic collagen networks
a. Collagen networks containing no cells or A431 clusters fixed and immunostained for Fibronectin, with additional DAPI (DNA) and Phalloidin (F-Actin) staining. The Fibronectin panel represents a brightest point projection over 25μm in the z-axis to capture the network in the region directly under the cluster. Representative image from n=23 clusters, N=3 independent experiment. Scale bar: 50μm. b. Average radial linescan (median ± SD) of fibronectin intensity from the cluster center to the periphery from data represented in e. At all distances, p > 0.05 for a two-tailed t-test with μ0 = 1. c. Collagen networks with A431 clusters seeded on top were fixed and immunostained for Collagen-IV and Laminin, with additional DAPI (DNA) and Phalloidin (F-Actin) staining. Representative image from n=5 clusters, N=1 independent experiments. Scale bar: 50μm. d. Fluorescent TAMRA-collagen networks with A431 clusters plated on top were fixed and immunostained for Collagen-I. Panels reflect single confocal slices at different z-steps. Colocalization between the TAMRA-Collagen and Collagen-I Antibody signal was R = 0.895 ± 0.040 (mean ± SD; p < 0.001) from n=15 clusters, N=2 independent experiments. Scale bar: 50μm. e. A431 cluster plated on a collagen network, fixed and immunostained with a Collagen-I antibody plus Phalloidin and imaged by two-photon microscopy. Representative image from n=9 clusters, N=2 experiments. Horizontal scale bars: 50μm, 25μm. Vertical scale bar: 10μm.
