Human interferon gamma ifn γ

Human prostate cancer cell lines PC3 and DU145 were obtained from ATCC (Manassas, VA, USA) and grown in a monolayer under standard culture conditions, 5% CO2 in a 37 °C incubator, in RPMI 1640 (Corning, Corning, New York, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cell identity was confirmed by short tandem repeat (STR) typing. Cells were tested for mycoplasma by PCR. For flow cytometry studies, cells were plated at a density of ~ 5,000/cm² into 6-well tissue culture plates or T-175 tissue culture flasks (for qPCR and RNA-Seq) and were allowed to adhere for 24 h. After 24 h, cells were treated with either 1 μM or 0.1 μM JQ1 (S7110, Selleckchem, Houston, TX, USA) and/or 100 units/mL of human interferon gamma (IFN-γ) (300–02, Pepro Tech, Rocky Hill, Connecticut, USA) for 48 h prior to harvest.

Human interleukin‐1‐beta (IL‐1β), tumour necrosis factor‐alpha (ΤΝF‐α) and human IL‐8 were purchased from R&D Systems, Inc. Human interferon‐gamma (IFN‐γ) was purchased from PeproTech (Rocky Hill, NJ, USA). The NF‐κΒ inhibitor pyrrolidinethiocarbamate ammonium (PDTC) was purchased from TOCRIS Biosciences (Bristol, UK). Dexamethasone (DXM) was purchased from Sigma‐Aldrich (St Louis, MO, USA); bone morphogenetic protein 2 (BMP2), BMP4 and BMP7 were purchased from R&D Systems, Inc.

Primary cultures of human skeletal muscle cells were initiated from myoblasts of DMD patients (n = 4; age range: 12–15 years) and healthy subjects (n = 3; age range: 15–17 years), which were provided by the French Telethon Myobank-AFM. Myoblasts were grown in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS), 1% L-Glutamine, 1% non-essential amino acids and 1% penicillin-streptomycin (all from ThermoFisher-Scientific) at 37°C in 5% CO2. After reaching a density of 80-90%, the growth medium was replaced with a fusion medium, where 20% FBS was replaced by 2% horse serum, and differentiation was allowed to continue for 14 days (time required to obtain mature myotubes with characteristic elongated and multinucleated morphology). Medium was changed every other day. Cells were always used at passages between 4 and 10. At day 14, myotubes were pre-treated or not with MCC950 (10 μM) for 24 h, while being challenged or not with human recombinant TNFα (15 ng/mL) + human interferon gamma (IFNγ) (15 ng/mL) (both from PeproTech, New Jersey, USA) for the last 22 h and then stimulated with ATP (5mM) (Roche, Basel, Switzerland) for the last 2 h.