Primary rib chondrocytes were isolated from the frontal part of the rib cage of 7-day-old mice. Muscle, soft tissues, and mineralized rib and sternal bones were removed using fine tweezers and surgical scissors under a dissection microscope. Dissected costal cartilage rods were then incubated in a digestion medium containing Dulbecco modified Eagle medium (DMEM), 10% fetal calf serum (FCS), and 0.2% (approximately 600 U/ml) collagenase type II (Worthington) at 37 °C for 6 hours. Cells were released from the cartilage matrix by gentle pipetting, passed through nylon mesh strainers (BD Falcon), spun to remove collagenase, counted, plated at the concentration of 1 × 105 cells/ml in the DMEM medium containing 10% FCS and cultured overnight.
Total RNA, including microRNAs, was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research). For Western blot analysis, cells were lysed in 2x Laemmli sample buffer. Proteins were resolved in 4 – 20% gradient acrylamide/MOPS gels, transferred on nitrocellulose membrane and incubated with indicated antibodies (anti-Hif-1a antibody (NB100-449) from Novus Biologicals, anti-beta actin antibody (#4970S) and anti-Ybx1 antibody (#9744S) from Cell Signaling Technology). Uncropped blots are shown in Source Data.
Total RNA, including microRNAs, was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research). For Western blot analysis, cells were lysed in 2x Laemmli sample buffer. Proteins were resolved in 4 – 20% gradient acrylamide/MOPS gels, transferred on nitrocellulose membrane and incubated with indicated antibodies (anti-Hif-1a antibody (NB100-449) from Novus Biologicals, anti-beta actin antibody (#4970S) and anti-Ybx1 antibody (#9744S) from Cell Signaling Technology). Uncropped blots are shown in Source Data.