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Fig. 3 Impact of PORCN inhibition while simultaneously activating canonical or non-canonical Wnt pathway on chondrogenesis. (A) Schema of individual differentiation steps of chondrocytes evaluated during the study together with the general effect of WNTs on early chondrogenesis, where non-canonical signaling enhances differentiation of chondrocytes towards hypertrophy while canonical signaling behaves antagonistically and maintains cells in non-differentiated mesenchymal status. (B) Schema of experimental design and summary of results in challenging simultaneously canonical and non-canonical signaling in the mesenchymal cells. PORCN inhibitor LGK974 was applied on cells alone or in combination with WNT5a or WNT3a to evaluate the effect of PORCN inhibition on both signaling pathways. (C) ECM production was evaluated on micromasses established from chicken forelimb buds (stage HH20) that were cultivated with 1 μM LGK974 in combination with WNT3a (10 ng/mL) or WNT5a (10 ng/mL) for 6 d. Application of LGK974 rescued WNT3a inhibitory effect on chondrogenesis and enhanced ECM production in WNT5a treated cultures. Scale bar = 1 mm. (D) Graphs displaying the analyses of blue pixels in Alcian blue stained micromasses after 6 d of cultivation. A decreased amount of ECM was observed after WNT3a treatment, and the addition of LGK974 restored its production. WNT5a did not affect ECM production, and its level was comparable to control cultures. Simultaneous treatment of WNT5a and LGK974 significantly increased the amount of ECM. Data is displayed as a number of blue pixels. One-way ANOVA followed by post hoc analysis using Fisher’s LSD test was used for statistical analyses (n = 4; spots evaluated per cultivated conditions) performed on samples from three independent experiments (see Supplementary material for other repetitions). (E) Graphs of Axin2 expression analyses of micromass cultures after 6-d cultivations evaluated by qPCR. Four spots were seeded in each well, four wells were analyzed as biological replicates per cultivated conditions and each sample was run in three technical replicates in each qPCR reaction. The level of Axin2 expression was related to Hprt1 gene expression, and FC as the ratio to control micromasses are displayed in individual treatments. Two-tailed Student’s t-test or Welch’s t-test was used for statistical analyses performed on samples from three independent experiments (see Supplementary material for other repetitions). Abbrevations: ECM, extracellular matrix; FC, fold changes; PORCN, Porcupine.