Growth supplement

Human astrocytes were cultured at 37°C, 5% CO₂ in astrocyte medium supplemented with 2% fetal bovine serum, growth supplement, and penicillin/streptomycin all obtained from ScienCell Research Laboratories (Carlsbad, CA, United States). Cells were cultured until they reached ∼80% confluence before passaging. At each passage, astrocytes were trypsinized, counted, and the cumulative population doubling level (CPDL) was calculated as we have previously described (Bitto et al., 2010 (link)). Cells were treated every 2–3 days for up to a week with either 0.3% DMSO as a vehicle control or the HAART drug combinations of abacavir (ABC) 10 μM and lamivudine (3TC) 5 μM or ABC, 3TC, and ritonavir (RTV) 1 μM. For the long-term experiments, cells were treated for up to 4 weeks with either 0.2% dimethyl sulfoxide (DMSO) as a vehicle control or the combinations of ABC 3 μM, 3TC 1.9 μM, atazanavir (ATV) 50 nM, and RTV 100 nM; or tenofovir (TDF) 100 nM, emtricitabine (FTC) 1.2 μM, ATV, and RTV; or TDF, FTC, and efavirenz (EFV) 125 nM. All HAART drugs were provided by the NIH AIDS Reagent Program.

Human mesenchymal stem cells with reverse telomerase transcriptase (hMSC-TERT) [34 (link)] were cultured in Eagle’s minimal essential medium (EMEM; PAA, Pasching, Austria) supplemented with 10% foetal bovine serum (FBS; PAA), 100 U/mL penicillin and 100 μg/mL streptomycin (both Biochrom, Berlin, Germany) as previously described [35 (link)]. Human NP cells (male, foetal, 20 weeks old) were obtained from ScienCell (Carlsbad, CA, USA) and expanded in Human Nucleus Pulposus Cell Medium containing 2% FBS and 1% growth supplement (ScienCell). Human chondrocytes (male, 65 years old) were obtained from Provitro (Berlin, Germany) and cultured in Chondrocyte Growth Medium containing 10% FBS (Provitro). All three cell types were cultured in different culture formats (25-300 cm² flasks) in a humidified incubator at 37°C with a 5% CO₂ atmosphere. Cells were passaged at 80% confluency and used during passages 69–75 (hMSC-TERT), 2-8 (NP cells) and 5-7 (chondrocytes). Cell morphology was analysed using a wide-field microscope.

Human preadipocytes from subcutaneous adipose tissue before passage 5 were thawed and maintained in growth medium (PAM; ScienCell Research Laboratories, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (ScienCell Research Laboratories), 1% growth supplement (ScienCell Research Laboratories), and 1% penicillin/streptomycin solution (ScienCell Research Laboratories). After reaching confluence and waiting another 1–2 days, the preadipocytes were exposed to serum-free induction medium containing 0.5 mM 3-isobutyl-L-methylxanthine (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 100 nM insulin (Sigma-Aldrich), 1 μM rosiglitazone (Sigma-Aldrich), and 1% penicillin/streptomycin solution for 4 days. Subsequently, the medium was replaced with the maintenance medium including DMEM/F-12 (Gibco), 100 nM insulin (Sigma-Aldrich), and 1% penicillin/streptomycin solution (Gibco) until the adipocytes were fully mature with and accumulated lipid droplet. The differentiated adipocytes were collected on days 4 and 8 for further detection.
To access FOXC2-AS1 expression level stimulated by forskolin (Fsk), differentiated adipocytes on days 4 and 8 were incubated in serum-free DMEM/F-12 medium for 6 h followed by stimulation with Fsk at a final concentration of 10 µM in DMEM/F-12 medium for additional 4–6 h to induce the thermogenic process in the adipocytes.

FreeStyle 293F suspension cells (Thermo Fisher Scientific) derived from human embryonic kidney cells were cultured in FreeStyle 293 Expression medium with 1% penicillin/streptomycin at 37°C and 8% CO₂ and were maintained at 0.15–1.2 x 10⁶ cells/ml on a cell shaker at 135 rpm. The HPMEC-ST1.6r line was kindly donated by Dr. J.C. Kirkpatrick at Johannes Gutenberg University, Germany, and was grown using the EGM-2 bullet kit (Clonetics, Lonza) and maintained as previously described [8 (link)]. HBMEC were donated by Dr. Ana Rodriguez at New York University and maintained using endothelial cell medium with growth supplement (ScienCell Research Labs).