Fig. 5

The effect of PORCN inhibitors on the growth of cartilaginous 3D cultures. (A) Schema of experimental design of 3D cultures. The mesenchymal cells separated from chicken embryonic limbs (stage HH26) were seeded as hanging drops or in PDMS microwell platforms. (B) LGK974 treatment (1 μM) increased the size of 3D hanging drop cultures following 14 d of cultivation. Microscopic sections stained by Alcian blue exhibit enlargement of organoids as well as hypertrophic chondrocytes. Scale bars: macroscopic view = 1 mm, sections = 100 μm. (B´) Immunohistochemistry labeling of cytoplasmatic membranes by Na+/K+-ATPase to visualize cell outlines. Scale bar = 100 μm. (B´´) graphs displaying organoids cultured with Wnt inhibitors for 11 d, where C59 (1 μM) or LGK974 (1 μM) treatment exhibited increased size of organoids, while PF670462 (1 μM) treatment did not significantly change their size. Data are displayed as the number of pixels of the organoid area in Wnt inhibitor-treated samples relative to the size of organoids cultivated in medium (n = 8; organoids evaluated per cultivated conditions). Two-tailed Student’s t-test or Welch’s t-test was used for the statistical analyses. (B´´´) Cell area analyzed in ImageJ using Na+/K+-ATPase labeled cells cultivated as hanging drops. LGK974 (1 μM) treated cultures exhibited statistically significant enlargement of the cell surface compared to cells grown in the medium. The data represent the cell area (in μm2) of PORCN inhibitor-treated samples relative to the cell area of organoids grown exclusively in the medium (n = 20; cells evaluated per cultivated conditions). Two-tailed Student’s t-test or Welch’s t-test was used for the statistical analyses performed on samples from three independent experiments (refer to the Supplementary material for 2 additional repetitions of the experiments). Abbrevation: PORCN, Porcupine.