A9205

Mouse C2C12 myoblasts were purchased from American Type Culture Collection (ATCC, no: CRL-1772) and used until passage 8. Myoblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose concentration, supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Long Island, NY, USA) and 1% antibiotic (50 IU/mL penicillin, 50 μg/mL streptomycin) and maintained in a humidified incubator at 37 °C in an atmosphere of 5% CO₂. Cells were seeded in 6-well plates, 100,000 cells per well, and when they reached 100% confluence, they were incubated with DMEM supplemented with 2% horse serum for 5 days to induce myotubes differentiation. PA (Palmitic acid, P5582, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol 100% and diluted in DMEM supplemented with 10% FBS or with 2% horse serum (Life Technologies) depending on the assay, containing 2% fatty acid-free bovine serum albumin (BSA) (A9205, Sigma-Aldrich) to reach desired fatty acid concentrations [24 (link)]. For the experiments, we used undifferentiated cells treated with 100 μM and 150 μM PA containing 2% BSA for 1 to 5 days or differentiated cells for 5 days and treated with PA for the subsequent 48 h (7-day differentiated cells). In both experiments, fresh media was provided every 24 h. A control group containing vehicle (DMEM, 2% BSA, and 0.19% ethanol) was also included in all experiments.

The human ESC line H9 (WiCell) was routinely cultured in commercial E8 medium (Cellapy, China) [17 (link)] (DMEM/F12 buffered by NaHCO₃, insulin, transferrin, sodium selenium, Vc, TGF-β1, and FGF-basic) on plates coated with the extracellular matrix Matrigel (354277, Corning). In some experiments, TGF-β1 and FGF-basic were removed from the E8 medium, resulting in the E6 medium. For passaging, cells were dissociated by 0.5 mM EDTA (GENOM, China) at 37 °C for approximately 5 min. Mediums were refreshed daily. Cells were maintained in an incubator at 37 °C and 5% CO₂.
For TSC induction, ESCs were passaged and seeded into six-well plates precoated with Matrigel at a density of 3 × 10⁵ cells/well. Overnight, the medium was removed and the cells were washed twice with PBS. Then, the induction medium was added to the wells. After 6 days, the cells were harvested for analyses. The induction duration may be shorter than 6 days as indicated in the figures. The components of the induction medium were as follows [9 (link)]: DMEM/F12 (11320033, Gibco), 0.3% BSA (A9205, Sigma), ITS-X (51500056, Gibco), 0.2% FBS (10099141C, Gibco), 10 μM Vc, 0.5 μM A83-01, 2 μM CHIR99021, 50 ng/ml EGF, 5 μM Y27632, and 10 ng/ml BMP4.

The PFA fixation and immunostaining processes were performed according to our previously reported methods [6 (link)]. After drug stimulation and washout, cells were fixed with 2% PFA for 15 min at 37 °C then washed twice with PBS. After blocking the cells with blocking buffer [1× PBS Tween-20 (28,352; Thermo Fisher Scientific) containing 1.5% (v/v) bovine serum albumin (A9205; Sigma-Aldrich)] for 30 min at room temperature (22–25 °C), the cells were incubated with a polyclonal antibody against rabbit alpha-actinin (AB90776; Abcam, Cambridge, UK), diluted with blocking buffer to a final concentration of 2 μg/mL, at 4 °C for 18 h. To analyze YAP1 nuclear localization, we used a rabbit monoclonal antibody against active YAP1 (AB205270; Abcam), diluted with blocking buffer to a final concentration of 0.2 μg/mL, at 4 °C for 18 h. After discarding the primary antibody solution, cells were washed twice with PBS and incubated with a secondary antibody solution, including goat anti-rabbit IgG (H + L) secondary antibody (A11034, Alexa Fluor 488, 1:400) and Hoechst 33342 (1:800; Thermo Fisher) at 4 °C for 1 h. Finally, cells were washed twice with PBS, and each well was filled with PBS before performing high-content analysis (HCA).