Digoxin

For Real-Time quantitative PCR, primary chondrosarcoma cells were treated with 0.5μM Digoxin (Cayman Chemical, 22266), or DMSO control for 5 days in culture and cells were lysed and RNA was extracted using Norgen Biotek Corp Single Cell RNA Isolation Kit (Norgen Biotek Corp, 51800). 1000ng of RNA was synthesized into cDNA using BioRad iScript (BioRad, 1708890) and diluted to a final concentration of 5 ng/μL of cDNA. The following forward and reverse primers indicated (Table S3) for each gene, mRNA transcript, was designed using NCBI’s PrimerBLAST for Homo saipan (human) and Mus musculus (mouse). SsoAdvanced Universal SYBR Green Supermix (BioRad, 1725272) was used to drive amplification. 2μL of cDNA (10ng) and 5μL of SYBR Green Mix, 0.4μM of forward primer, 0.4μM of reverse primer to a final volume of 10μL per PCR reaction was used. Relative gene expression was compared to DMSO control treated group for individual patient cell lines which were normalized to 1 was calculated and normalized to beta-actin using the 2^−ΔΔCt method.

Idh1-KI R132Q and Col2a1Cre-ERT mice were crossed and injected with 20 mg/kg tamoxifen (Sigma-Aldrich, 579002) and 10 mg/kg progesterone (Thermo Fisher Scientific, AC225650050) at embryo day 12.5 (E12.5). Mice were sacrificed at E16.5. Both hindlimbs of embryos were placed on nitrocellulose membranes (Thermo Fisher Scientific, 88024) for adhesion and treated with 0.5μM Digoxin (Cayman Chemical, 22266) or DMSO control for 5 days. Digoxin and DMSO were dissolved in the following media: 500mL aMEM (Thermo Fisher Scientific, 12571063), 5mL of 10,000 U/mL penicillin streptomycin (P/S) (Thermo Fisher Scientific, 15140122), 25mg ascorbic acid (Thermo Fisher Scientific, AC105021000), 108mg B-glycerophosphate (Cayman Chemical, 14405), 1g BSA (Thermo Fisher Scientific, 11020021). After 5-day treatment, hindlimbs were fixed with 10% formalin and placed overnight at 4°C. Tissues were washed with 70% EtOH and processed for paraffin embedding and sectioning.