Fig 2: Characterization of 3D-cultured ASC spheroids. A Representative light microscopy images of 2D-ASCs (a) and 3D-ASCs at 72 h (b) or at 7 days (c) of sphere culturing. B Representative phase contrast microscopy image (a) and fluorescence image (b) showing 3D-ASC morphology. Arrows indicate elongated cells at the edge of an ASC spheroid. C Representative fluorescence images showing the expression of the mesenchymal marker Vimentin (green; a, b), the ASC-related markers CD29 (green; c, d) and CD166 (green; e, f), and the pluripotency genes cMyc (green; h, i) and Sox2 (red; j, l) in 2D- and 3D-ASCs. D Representative fluorescence images showing organization of the F-actin cytoskeleton (red; a, b), expression of E-cadherin (green; c, d) and presence of the extracellular matrix protein Col1a1 (green; e, f) in 2D- and 3D-ASCs. DAPI, 4’,6-diamidino-2- phenylindole. Scale bars, 100 μm. E Western blot analysis of Vimentin, cMyc, Sox2 and E-cadherin protein expression in 2D- and 3D-ASCs. HSP90 was used as internal control. The intensity of the bands was evaluated by densitometric analysis, the values were normalized and reported in graph as fold increase with respect to 2D-ASCs. *P < 0.05, **P < 0.01 vs 2D-ASCs