Fig 1: YAP regulates FA formation and cell stiffness in response to cell area in AD-MSCs.(a) Left: confocal analysis of individual cells grown onto 300, 1,024, 2,025, 10,000 µm2 micropatterns coated with either fibronectin or N-cadherin. Right: three-dimensional (3D) reconstruction of individual cells grown onto 10,000 µm2. Cells were stained with: anti-vinculin (green) Alexa Fluor 546 Phalloidin and 4',6-diamidino-2-phenylindole (DAPI). Cells were infected with viral particles encoding for either control short hairpin RNA (CTR), short-hairpin RNA targeting YAP or TAZ (shYAP, shTAZ, respectively). (b) Graph: Young's modulus analysis of single AD-MSCs, shYAP and shTAZ onto micropatterns coated with FN or PLL. Values are shown as median±min/max (n=24, 3 technical replicates, Kruskal–Wallis test followed by Dunn's test, ****P<0.0001). (c) Venn diagram showing the overlap between genes significantly regulated in shYAP and shTAZ cells as obtained by quantitative PCR arrays (see Supplementary Fig. 3a). (d) Schematic structure of YAP (S127A, ?PDZ) and TAZ (S89A, ?304) mutants used for the transfections. (e) Confocal images of focal adhesion distribution, as stained by anti-vinculin (red) and DAPI in AD-MSCs (left) and CAL51 (centre) transfected with the indicated YAP and TAZ mutants. Right: Representative image analysis of vinculin expression in AD-MSCs and CAL51 transfected or not with S127A YAP mutant. Transfected AD-MSCs were stained with anti-vinculin antibody (red). Transfected CAL51 cells were stained with anti-vinculin (red), Alexa Fluor 647 Phalloidin (white) and DAPI. Transfected cells are identified by GFP expression and highlighted by dashed lines.