Quantitative and statistical analyses of leader bleb area, cortex tension and intracellular pressure for each condition in A375 cells. (Sheets 1–6) Quantitative and statistical analyses of leader bleb area (Sheets 1 and 2, expressed in μm2) cortex tension (Sheets 3 and 5, expressed in pN/µm) and intracellular pressure (Sheets 4 and 6, expressed in Pa) for human melanoma A375 cells treated with non-targeting siRNA (non-targeting) or depleted of Eps8 using an siRNA specific for human Eps8 (hEps8 siRNA), rescued with or over-expressing (OE) Emerald-tagged wild type mouse Eps8 (mEps8 WT) or the following mutants: mEps8 Δbund (bundling defective, L757A/K759A), mEps8 Δcap (capping defective, V689D/L693D) and mEps8 SATA (Erk phosphorylation deficient, S624A/T628A), or EGFP-tagged human α-actinin, or treated with 50 µM blebbistatin to inhibit myosin II or 10 µM U0126 to inhibit MEK/Erk. (Sheets 1, 2) Cells were confined between uncoated glass and an agar pad, leader bleb area is expressed as percent of cell body area. In (Sheet 1), cells were depleted of Eps8 and rescued with WT and mutants of Eps8, in (Sheet 2), cells were over-expressing WT or mutant Eps8. (Sheets 3–6) Cells were plated on uncoated glass, and where noted, treated with 50 µM blebbistatin (5 min) to inhibit myosin II or 10 µM U0126 (30 min) prior to atomic force microscopy analysis. (Sheets 3, 5) Cortex tension (expressed in pN/µm) in cells (Sheet 3) depleted of and rescued with WT Eps8, or (Sheet 5) over-expressing WT Eps8 or mutants. (Sheets 4, 6) Intracellular pressure (expressed in Pa) in cells (Sheet 4) depleted of and rescued with WT Eps8, or (Sheet 6) over-expressing WT Eps8 and mutants.