Spheroid invasion assay time course. 4T1 spheroids expressing membrane-tethered Venus were embedded in an ECM scaffold (80% collagen type I, 20% Matrigel), then bathed in serum-containing medium with 5 uM Rhobo6. Volumes of 553 um x 553 um x 162 um were acquired once per hour over 2 days using 2P microscopy. Maximum intensity projections over 12 um are shown. Yellow boxes represent examples of non-invading (1) and invading (2) regions shown in b .
