Fig. 3. The effect of cell density, cell geometry, and ECM stiffness on YAP/TAZ localization and cell proliferation. High cell Density, small cell geometry, and soft ECM lead to YAP/TAZ inactivation, and growth arrest. (A) Cell area in the three seeding conditions. Cells plated at different densities display increasingly smaller cell-substrate adhesion areas. Cells seeded to obtain sparse cells and confluent or dense monolayers. After 2 days, cells fixed for immunofluorescence with anti-E-cadherin antibody (αE-CAD) to visualize the formation of cell-cell contacts. TOTO3 is a nuclear counterstain. Scale bar, 20 μm. (Right) Average cell area in the three seeding conditions. (B) Cell proliferation in the three seeding conditions which measured as the percentage of BrdU-positive cells. Cell plated as in (A); after 2 days, cells were incubated with BrdU to label cells undergoing DNA duplication. Then cells were fixed and processed for anti-BrdU immunofluorescence (αBRDU). (Right) Quantitation of cell proliferation in the three seeding conditions. (C) Nuclear/cytoplasmic localization of YAP/TAZ in the three seeding conditions. Cells plated as in (A) and stained for immunofluorescence with anti-YAP/TAZ antibody (αYAP/TAZ). TOTO3 is a nuclear counterstain. Scale bar, 20 μm. (Right) The proportion of cells displaying preferential nuclear YAP/TAZ localization (N, black), even distribution of YAP/TAZ in nucleus and cytoplasm (N/C, gray), or cytoplasmic YAP/TAZ (C, white). (D) Effects of restricting cell-substrate adhesion area (Cell geometry) and (G) ECM substrate stiffness on YAP/TAZ localization and cell proliferation. In (D), immunofluorescence images of YAP/TAZ with anti-YAP/TAZ antibody (αYAP/TAZ). DAPI is a nuclear counterstain. Scale bar, 20 μm. (E) YAP/TAZ nuclear/cytoplasmic localization and (F) percentage of cell proliferation which processed for BrdU. In (G), Immunofluorescence images of YAP/TAZ in plated cells on fibronectin-coated stiff (plastic) and soft (acrylamide hydrogels of 0.7 kPa) substrates using anti-YAP/TAZ antibody (αYAP/TAZ). TOTO3 is a nuclear counterstain. Scale bar, 20 μm. (H) YAP/TAZ nuclear/cytoplasmic localization and (I) percentage of cell proliferation which processed for BrdU. Down: Cells seeded as individual cells plated on fibronectin-coated glass (large) or square microprinted fibronectin islands of 300 μm2 (small). (Reprinted with permission, Cell Press, for citation see [53]).