Total RNA was extracted from cultured primary human chondrocytes using a PureLink RNA Mini Kit (Ambion, Cat#12183018A) and 2-mercaptoethanol (Thermo Fisher Scientific, Cat#31350010). To obtain complementary DNA (cDNA), 50 ng RNA was reverse-transcribed via a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat#4368814) using a thermal cycler (ProFlex, Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. For qRT-PCR analysis, all the genes were amplified using TaqMan® Gene Expression Assays for CHAD (Cat#4331182, Hs00154382_m1), HIF1α (Cat#4331182, Hs00153153_m1), COL2A1 (Cat#4331182, Hs00264051_m1), and internal control genes (housekeeping genes: actin beta (ACTB, Cat#4331182, Hs01060665_g1) in accordance with the manufacturer’s protocol [11 (link), 15 (link), 16 ]. Each gene was amplified using an RT-PCR reaction mix prepared with 1 μl TaqMan Gene Expression Assay, 10 ml of TaqMan Gene Expression Master Mix (Cat. #4369016), 4 ml of cDNA template, and UltraPure DNase/ RNase-Free distilled water (Cat. #10977035) on MicroAmp Fast Optical 96-well reaction plates (Cat. #4346906). The Applied Biosystems 7300/7500 RT-PCR system was used with the following reaction protocol: maintained at 50°C for 2 min, maintained at 95°C for 10 min, and alternating between 95°C for 15 s and at 60°C for 1 min and 40 cycles.