10 cm cell culture dishes

The RAW264.7 macrophage cell line was sourced from the American Type Culture Collection (ATCC), located in Manassas, VA, USA. These cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco brand) which was fortified with 10% fetal bovine serum (Gibco) and an additional 1% penicillin/streptomycin antibiotic blend (Gibco). The culture conditions maintained were at a steady temperature of 37°C under a humidified atmosphere containing 5% CO₂. RAW264.7 cells were seeded onto 10 cm cell culture dishes (Corning Inc., New York, USA) and the medium was refreshed every two days to ensure optimal growth. Cell passage was carried out once the cells achieved approximately 90% confluence. This process involved discarding the used culture medium, followed by rinsing the adherent cells twice with PBS. Subsequently, the cells were detached and re-suspended in fresh medium at a ratio of 1:3 for further propagation. In subsequent experiments, RAW264.7 cells were treated with 1 μg/ml LPS (L3024, Sigma, MO, USA) for 24 h as described in our previous publication [59 (link)]. Erastin (10 μM) (Selleck, USA), rapamycin (20 μM) (Selleck), 4-P-PDOT (10 μM) (Sigma) and luzindole (10 μM) (Sigma) were treated 3 h before LPS stimulation. MT (500 μM) (Sigma) was treated 1 h after LPS stimulation. All cells were verified to be free of mycoplasma.

Immortalized mouse mammary fibroblasts were generated in Dr. Harold Moses laboratory (Vanderbilt University), have been used in previous publications [46 (link)–48 (link)], and gifted to us. Primary mouse mammary fibroblasts were isolated from mouse mammary gland by FACS sorting as described below. Fibroblasts were grown in T-75 flasks (Fisher Scientific, USA) in DMEM medium (Gibco, USA) supplemented with 10% FBS, Antibiotic-Antimycotic (Sigma-Aldrich). For protein assessment, fibroblasts were grown in 10cm cell culture dishes (Corning, USA). When cells achieved 90–95% confluency, they were treated with TGFβ, 1ng/ml (R&D System, USA) and 0.01, 0.1, 1, 10, 100 uM NECA (5′-N-Ethylcarboxamido adenosine, Sigma-Aldrich, USA). Following inhibitors were used: for inhibition of A2a adenosine receptors, 1 uM SCH58261(Tocris, USA); for A2b receptors– 1 uM PSB603 (Tocris, USA); for PLC inhibition– 1 uM U73122 (Tocris, USA); for PKA inhibition—1 uM H89 (Tocris, USA); and for activation of adenylate cyclase– 10 uM forskolin (Sigma-Aldrich, USA). Human fibroblast cell lines IMR-90 (CCL-186), WS-1 (CRL-1502), WPMY-1 (CRL-2584), hTERT SMC PM151T (“hTERT” CRL-3291) and BJ (CRL-2522) were purchased from ATCC and cultured according to the manufacturer’s protocol.

The myoblasts were cultured in six-well plates (Corning, New York, NY, USA) before transfection. The goat myoblasts were used for cell transfection when they reached ~60% confluence. Before 24 h of transfection, 293T cells were inoculated into 10 cm cell-culture dishes (Corning, USA) and co-transfected with recombinant plasmids and the packaging plasmids, pMD2G and psPAX2, via Lipofectamine^TM 3000 transfection reagent (Thermo Fisher Scientific). The existing medium was replaced with fresh complete medium after 6 h of transfection. Cell cultures were collected after 48 h and 72 h transfection, passed through a 0.22-micrometer filter and centrifuged at 72,000× g and 4 °C for 2 h. The supernatant was discarded and the pellet was resuspended in 500 μL fresh culture medium to obtain the viral fluid. Successful infection of the goat myoblast by the viral fluid was observed under a fluorescent microscope (Nikon, Tokyo, Japan) and appeared as green fluorescence. When the cells reached 90% confluence, they were subjected to cell-differentiation medium containing 2% (v/v) horse serum. That time point was recorded as day 0 of differentiation. The cell-differentiation medium was changed every 2 d. Total RNA and protein were collected from the cells at days 0 and 6 of differentiation. Cell morphology and myotube appearance were observed under a microscope.