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Figure S1. ROCK mediates p53-dependent regulation of the subcellular distribution of integrin β1 and ezrin in rounded migrating cells cultured in 3D Matrigel. Representative integrin β1 (top) and ezrin (bottom) fluorescent staining of MEFs and p53^-/- MEFs alone, treated with Y27632, or transfected with the plasmids p53 H273 or ROCK-Δ1 as indicated. Insets show costaining of F-actin (red), GFP labeling (green) to identify transfected cells, and ezrin or integrin β1 (blue) as indicated. Cells were plated in Matrigel, and a gradient of serum growth factors was established. Cells were fixed and prepared for immunofluorescence as described in Materials and methods. Arrows indicate the direction of cell movement in Matrigel. Bars, 10 µm. Treatment of p53^-/- MEFs with Y27632 inhibits dynamic blebbing and converts cells from a spherical to an elongated morphology. This transition is accompanied with the redistribution of integrin β1 and ezrin because their clustered distribution is lost in p53^-/- MEFs treated with Y27632. On the contrary, the expression of p53 H273 and ROCK-Δ1 converts MEFs to an elongated shape and leads to the loss of the uniform staining of integrin β1 and ezrin throughout the cell. Instead, both show an asymmetrical clustered localization in the moving front of cells.