Gene Expression Analysis of TGFβ3-Treated Pellets

For pellets that were cultured with TGFβ3 supplementation, we ground the pellets with Molecular Grinding Resin (G-Biosciences, Saint Louis, MO, United States, 786-138PR) extracted total RNA using Tri-Reagent (Sigma-Aldrich). Due to the considerably smaller size of the pellets cultured without TGFβ3 supplementation, we ground the pellets using the same grinding resin as above and used the RNeasy mini kit (Qiagen, Toronto, ON, Canada); 100 ng of the isolated total RNA was reverse transcribed into cDNA for qPCR analysis using the gene-specific primers listed in Supplementary Table 1. Expression of genes of interest was normalized to the mean expression level of reference genes YWHAZ, β-actin, and B2M (Foldager et al., 2009 (link); Munir et al., 2014 (link)) and presented using the 2ΔCt method (Schmittgen and Livak, 2008 (link)).