Fig. 7

Determination of PORCN inhibitor’s effect on cell proliferation versus differentiation in early stages of cultivation. (A) Schema of individual chondrocyte differentiation steps in micromass cultures evaluated by qPCR. (B) Graphs of gene expression analyses of micromass cultures established from chicken limb buds (stage HH20) after 1, 2, and 3 d of cultivations, which were evaluated by qPCR. The level of CCND1 expression was reduced in the 1-d or 2-d cultivations with both PORCN inhibitors—C59 and LGK974 (1 μM), with no change observed in the expression at 3 d compared to control cultures. Mmp13 expression was significantly increased in 2-d micromass cultures after PORCN inhibition, and this trend was even more evident in 3-d micromasses. The gene of interest expression level was related to Hprt1 gene expression, and FC as ratio to control micromasses are shown on graphs. Two-tailed Student’s t-test, Welch’s t-test or Mann–Whitney test was used for statistical analyses performed on samples from three independent experiments (refer to the Supplementary material for 2 additional repetitions of the experiments). Abbrevations: FC, fold changes; PORCN, Porcupine.