Transdifferentiation Assay for Osteogenesis

The transdifferentiation assay was performed as described before.^{41 (link)} Briefly, ATDC5 cells were cultured in chondrogenic differentiation medium for 7 days and thereafter cultured in osteogenic differentiation medium containing 50 ng/mL recombinant human Ctgf (PeproTech Inc., Rocky Hill, NJ, USA). Cells were harvested after 24 and 48 h of osteogenic differentiation, respectively. RNA from cell lysates was isolated using the RNeasy Mini Kit and cDNA was generated using Omniscript Reverse Transcriptase (both Qiagen). qRT-PCR expression analysis was performed using Platinum™ SYBR™ Green qPCR SuperMix-UDG and ROX™ Reference Dye (both Thermo Fisher Scientific). The primer sequences of the analyzed genes are shown in Table S1. B2m expression was used as the reference housekeeping gene. Data analysis was performed according to the delta-delta comparative threshold cycle (2^-ΔΔCT) method, and results are shown as fold-change expression values relative to controls.