Selenious acid

To induce chondrogenic differentiation, cells were trypsinized, transferred into a 15-mL polypropylene tube, centrifuged at 1000 rpm for 5 min to form a pelleted micromass at the bottom of the tube, and then treated with chondrogenic medium [high-glucose DMEM supplemented with 0.1 M dexamethasone, 50 g/mL AsA (Sigma-Aldrich), 100 g/mL sodium pyruvate (Sigma-Aldrich), 40 g/mL proline (Sigma-Aldrich), 10 ng/mL TGF-β1 (R&D systems, Minneapolis, MN, USA), and 50 mg/mL ITSpremix (Gibco/Invitrogen), 6.25 μg/mL insulin, 6.25 μg/mL transferrin (Sigma-Aldrich), 6.25 ng/mL selenious acid (Sigma-Aldrich), 1.25 mg/mL bovine serum albumin (BSA), and 5.35 mg/mL linoleic acid (Sigma-Aldrich)] and control medium (high-glucose DMEM). Medium changes were carried out twice weekly, and chondrogenesis was assessed at weekly intervals. After 2–4 weeks of culture, the cells were washed twice with PBS, fixed in 4% paraformaldehyde, and visualized either by staining with Alcian Blue (Sigma-Aldrich) [11 (link)].

Peptide DJK-5 (VQWRAIRVRVIR-NH₂; all D amino acids as indicated in italics) was synthesized by CPC Scientific (Sunnyvale, CA, USA) using solid-phase 9-fluorenylmethoxy carbonyl chemistry and purified to ~95% using reverse-phase high-performance liquid chromatography. Peptide identity was confirmed by mass spectrometry. N/TERT cell and skin culture medium including Keratinocyte-SFM medium, Dulbecco’s Modified Eagle Medium (DMEM, high glucose, GlutaMAX™ Supplement, pyruvate), and Ham’s F-12 Nutrient Mix were purchased from ThermoFisher Scientific (Waltham, MA, USA). CnT-Prime 3D Barrier Medium was obtained from CELLnTEC Advanced Cell Systems AG (Zurich, Switzerland). DermaLife K Keratinocyte Medium Complete Kit was purchased from Lifeline Cell Technology (Oceanside, CA, USA). Supplements for skin culture medium including isoproterenol, hydrocortisone, bovine insulin, selenious acid, l-serine, l-carnitine, bovine serum albumin, palmitic acid, linoleic acid, and arachidonic acid were obtained from Sigma-Aldrich (St. Louis, MI, USA). Antibiotic controls (gentamicin and fusidic acid) and 10% neutral-buffered formalin solution were also purchased from Sigma-Aldrich. Reagents for bacterial culture and resuspension including tryptic soy broth (TSB), Luria broth, d-glucose, and phosphate-buffered saline (PBS) were purchased from ThermoFisher Scientific.