Its liquid media supplement

The mouse hepatocyte cell line AML12 was treated with CoCl₂ (Sigma-Aldrich, St. Louis, USA) to establish the cell hypoxia model as described previously [29 (link)]. AML12 cells were cultured in DMEM/F12 (Hyclone, Logan, USA) containing 10% fetal bovine serum (FBS), ITS liquid media supplement (Sigma-Aldrich, St. Louis, USA), and 40 ng/mL dexamethasone (Solarbio, Beijing, China) at 37°C with 5% CO₂. The cells divided into four treatment groups. These were the control, ORY (240 μg/mL), CoCl₂ (300 μM), and CoCl₂+ORY (240 μg/mL ORY was added at the same time with CoCl₂ (300 μM)) groups [30 (link)]. Levels of ROS in AML12 were determined by DCFH-DA ROS assay kit (Beyotime, Shanghai, China) according to the manufacturer's instruction. The mouse hepatocyte cell line AML12 was treated with tunicamycin (TM) (Solarbio, Beijing, China) as described previously [31 (link)]. These were the control, ORY (240 μg/mL), TM (10 μg/mL), and TM+ORY. The cells were pretreated with γ-oryzanol for 12 h, followed by treatment with 10 μg/mL tunicamycin for an additional 24 h.

Validated U2OS, HeLa, CAL72, HCT116, and SAOS2 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The MG63 cell line was purchased from CLS Cell Lines Service (Eppelheim, Germany). U2OS, HeLa, and CAL72 cells were maintained in DMEM supplemented with 10% FCS, 2 mM l-glutamine, 1% antibiotics. For CAL72, 1X ITS liquid media supplement (Sigma-Aldrich) was added to this medium. The other three cell lines studied were cultured in 90% McCoy’s 5A supplemented with 10% FCS, 2 mM l-glutamine, and 1% antibiotics (HCT116), in 85% McCoy’s 5A supplemented with 20% FCS (SAOS2) or in DMEM/Ham’s F12 medium supplemented with 5% FCS, 2 mM l-glutamine, and 1% antibiotics (MG63). An U2OS cell line for inducible expression of ATRX (referred to here as U2OS^ATRX-2) was kindly provided by Richard Gibbons (University of Oxford, UK) and has been described previously (8 (link)). The U2OS^ATRX-2 cells were cultured in DMEM supplemented with 10% doxycycline-free FCS, 2 mM l-glutamine, 1% antibiotics, 0.5 μg/ml puromycin, 0.7 μg/ml G418.

The mouse ID8 IP2 cell line was developed in the lab of co-author Jill Slack-Davis and provided directly to the Kitajewski lab [29 (link)]. ID8 IP2 cells were cultured in DMEM with 10% FBS, 1% ITS Liquid Media Supplement (Sigma-Aldrich I3146), and 1% penicillin-streptomycin, and infected with lentiviral vector FUW-luciferase-mCherry [71 (link)]. Human ovarian cancer cell lines OVCAR3. OVSAHO, and OVCA429 were a kind gift of Dr. Joanna Burdette (University of Illinois Chicago), A2780 was a kind gift of Dr. Tian-Li Wang (Johns Hopkins), and SKOV3-IP1 was a kind gift of Dr. Olga Razorenova (University of California Irvine). OVCAR3, A2780, OVSAHO and SKOV3-IP1 were cultured in RPMI 1640 with 10% FBS and 1% penicillin-streptomycin, while OVCA429 was cultured in MEM with 10%FBS, 1% L-glutamine, 1% NEAA, 1% Sodium pyruvate and 1% penicillin-streptomycin. Mouse and human cells were lentivirally infected with virus derived from a pCCL vector encoding an HA-tagged Notch3 intracellular domain (codons 1664–2318) followed by an IRES-GFP (Notch3IC, Fig 1C) or empty pCCL vector. Five matched sets of Notch3IC and control ID8 IP2 lines were generated, identified as Sets #1-#5 throughout. Human cells were freshly infected prior to each experiment. Post-hoc testing suggests that all ID8 IP2 lines were mycoplasma positive throughout these experiments.

For explant culture, cartilage tissues were obtained from 5 OA knees in pairs from preserved areas and degenerated areas on the tibial plateaus, as described for the cartilage protein analysis. After being rinsed in sterilized PBS, each piece of cartilage tissue was diced into 30–50 cubes of 2–3 mm. The diced cartilage, or explants, were equally divided into two, which were placed onto respective wells of a 12-well plastic plate. These explants were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with ITS Liquid Media Supplement (Sigma Aldrich, St. Louis, MO, USA) and 25 µg/ml ascorbic acid overnight, and then the media was replaced with those containing Akt inhibitor IV (Sigma Aldrich; 5 µΜ in dimethyl sulfoxide [DMSO]) or DMSO alone (vehicle control). After 48 h of culture, the explants were recovered, embedded in OCT compound, snap frozen in liquid nitrogen and stored at -80 °C until analysis. Extraction of RNA from the explants was performed following a previously described method [3 (link)]. In brief, 20 µµ-thick cryosections were prepared from the explants embedded in OCT compound, which were immediately immersed in TRIzol (Thermo Fisher Scientific). RNA was first recovered from the TRIzol reagent in the aqueous phase, and then purified using the RNeasy Micro kit (Qiagen).

Cardiomyocytes CMs were isolated from adult mouse hearts (8–10 weeks of age) using a simplified Langendorff-free method^{78 (link)} and cultured in M199 (Sigma, M4530) supplemented with 0.1% BSA (supplier, number), 1X ITS Liquid Media Supplement (Sigma, I3146), 10 mmol/l BDM (Sigma, B0753), 1X Chemically Defined Lipid Concentrate (Fisher, 11548846) and penicillin–streptomycin (Thermo Fisher, 15140-122). When needed, adenovirus was administered to CMs in culture media immediately after removal of plating media. Medium was then replaced after 24 h with culture media together with 100 nM (Z)-4-OHT (Sigma, H7904) in ethanol and collected after 4 h for RNA extraction.

rMSCs were suspended in medium and then seeded into scaffolds at a density of 1 × 10⁷ cells/mL. The tri-copolymer scaffold/rMSCs constructs were placed in a culture plate for 24 h for cell adhesion, then either cultured in static condition for seven days or cultured in the self-designed bioreactor system up to 21 days. After 24 h for cell adhesion, all the constructs were cultured with chondrogenic medium. The chondrogenic medium contained LG-DMEM, 10% FBS, 1x ITS liquid media supplement (Sigma-Aldrich, St. Louis, MO, USA), 50 µg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 40 µg/mL proline (Sigma-Aldrich, St. Louis, MO, USA), 100 µg/mL sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), and 0.1 µM or 1.0 µM of KGN (Merck, Darmstadt, Germany). At each time period, 5 mL of the medium were sampled for GAGs quantification (n = 3) via dimethylmethylene blue (DMMB, Sigma-Aldrich, St. Louis, MO, USA) assay.