C3h10t1 2 clone 8

The murine multipotential stem cell line, C3H10T1/2 clone 8, was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Monolayer cultures were maintained in DMEM containing 10% fetal calf serum (FCS) (growth medium). To induce chondrogenesis, we used a miromass culture system, ITS  +  premix (insulin, transferrin, selenous acid, BSA, and linoleic acid; Corning, Corning, NY, USA) and bone morphogenetic protein (BMP)-2 (Peprotech, Rocky Hill, NJ, USA) using 10 µL drops of cells at 1 × 10⁷ cells/mL as described previously [56 (link)]. Micromass cultures were maintained in the growth medium with or without 100 ng/mL recombinant murine BMP-2 and P15-1 (17 µg/mL) for up to 6 days. The medium was changed every other day. Total RNA was isolated after 3 days of micromass cultures and analyzed for the mRNA levels of chondrocyte markers, Sox-9 and type II collagen, and the stem cell marker, type I collagen. In addition, Alcian blue staining (1% Alcian blue (Sigma-Aldrich, St. Louis, MO, USA) in 3% glacial acetic acid solution) of the micromasses was performed to determine the degree of chondrogenic differentiation.

HEK293T, Huh7, RKO, U2OS, 3T3-L1, C3H10T1/2 clone 8, HCT116, Ls174t, SW480, HT-29, DLD1, and Caco-2 were purchased from American Type Culture Collection (ATCC), YAPC and ST2 were obtained from DSMZ. HEK293T, YAPC, Huh7, SW480 and 3T3-L1 were grown in DMEM (Cat#11995-040, Invitrogen) with 10% FBS (Cat#1500-500, Seradigm) and penicillin/streptomycin (Cat#10378016, Invitrogen). ST2 and DLD1 were maintained in RPMI-1640 (Cat#22400-071, Invitrogen) with 10% FBS and penicillin/streptomycin. U2OS, HT-29 and HCT116 were grown in McCoy’s 5A (Cat#16600-082, Invitrogen) with 10% FBS and penicillin/streptomycin. RKO, Caco-2, Ls174t and C3H10T1/2 were maintained in MEM (Cat#11095-080, Invitrogen) with 10% FBS and penicillin/streptomycin. All cultures were grown at 37 °C in a 5% CO2 incubator. Cell lines were tested to be free of mycoplasma contamination, and were not subjected to extra authentication steps.
Plasmids were generated using standard recombinant DNA techniques. Site-specific mutagenesis was performed using Q5 Site-Directed Mutagenesis Kit (New England Biolabs). Primers used for cloning are shown in Supplementary Table 1.

Mouse embryonic fibroblasts (C3H10T1/2 clone 8 from American Type Culture Collection) were grown in basal eagle medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO₂ in air. For plating cells were harvested by 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) in 0.02% EDTA in PBS (w/o Ca²⁺, Mg²⁺).