Scc049

The O9-1 mouse cranial neural crest cell line was purchased from MilliporeSigma (SCC049) and cultured in complete ES cell medium containing 15% FBS and LIF (MilliporeSigma, ES-101-B) with 100 units/ml penicillin-streptomycin (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO₂ according to the manufacturer’s instructions. For osteogenic or chondrogenic differentiation, O9-1 cells were cultured with StemMACS OsteoDiff Media or StemMACS ChondroDiff Media (Milteny Biotec), respectively. Osteogenic differentiation and chondrogenic differentiation were evaluated by alizarin red or alcian blue staining as previously described [70 (link),73 (link)]. To knockdown Tmem2 expression in O9-1 cells, we used lentivirus-mediated shRNA transduction. Lentivirus particles expressing an shRNA that is validated to deplete mouse Tmem2 (Mission shRNA, TRC Clone ID: TRCN0000295501, MilliporeSigma) and control lentivirus particles expressing an shRNA that does not target any known genes (Mission shRNA, MilliporeSigma SHC005) were purchased from MilliporeSigma. Lentivirus particles were added to O9-1 cells cultured in growth media supplemented with 5 μg/ml polybrene and cultured for 2 days. Cells transduced with lentiviral shRNAs were selected and maintained in the presence of 10 μg/mL puromycin.

Plasmids (pcDNA3.1+/C-(K)-DYK) encoding full-length mouse Cdx1, Cdx2, and Cdx4 were obtained from Genscript. O9-1 mouse cranial neural crest cells (CNCC, SCC049, Millipore Sigma) were transfected with Cdx constructs using EndoFectin Max transfection reagent (GeneCopoeia, Rockville, MD, USA). The transfected cells were split after 48 h and selected for stable plasmid integration using G418 antibiotic (Gibco, ThermoFisher Scientific, Waltham, MA, USA) for 8–10 days. Resistant cell colonies were subcultured and overexpression ascertained by q-PCR analysis before use in the experiments.