FMNL2 localizes to AJs in a 3D model for nascent cell–cell adhesion formation. (A) Confocal images of MCF10A cells in 3D stained for F-actin after 1, 4, or 14 d. (B) 3D reconstructions of MCF10A cells expressing LifeAct-mCherry and E-Cadherin–GFP during cell–cell contact formation. Merged images show magnification (bar, 2 µm). (C) MCF10A cells grown in Matrigel and labeled as indicated. The asterisk marks the junctional area. (D) Expression of formins in MCF10A cells assessed by qPCR. (E) MCF10A cells grown in Matrigel were labeled as indicated. FMNL2 localizes to the AJ (asterisk). (F) MCF10A cells expressing FMNL2-GFP were labeled for E-Cadherin. (G) 3D reconstructions of a time series of MCF10A cells expressing LifeAct-mCherry and FMNL2-GFP. Merged images magnify the AJ area (bar, 2 µm). (H) Representative images of MCF10A cells expressing GFP or FMNL2ΔDAD-GFP stained for F-actin. Arrows illustrate line scans used for quantifications. (I) Corresponding line scan profiles to H. (J) Quantification of F-actin line scan profiles (GFP, n = 47; FMNL2ΔDAD-GFP, n = 66). *, P ≤ 0.05. Error bars indicate SEM.