2.6. H9c2 Culture and Treatment

The embryonic rat heart-derived cell line H9c2 was obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Gibco, Eggenstein, Germany) containing 2.25  g/L glucose medium supplemented with 10% fetal bovine serum (FBS), 100  U/mL of penicillin, and 100 mg/mL of streptomycin, at 37 °C with 5% CO₂ in humidified air. Cells between passages 4 and 8 were used for the experiments. For in vitro studies, stock solutions of 5 mM PA/10% fatty acid-free bovine serum albumin (BSA) were prepared and stored at −20 °C. Stock solutions were heated for 5 min at 65 °C and then cooled to room temperature before use. The fatty acid-BSA complex was added to the serum-containing cell culture medium to achieve a fatty acid concentration of 100 μM.

Commercial Nr4a1 siRNA was used to inhibit NR4A1 protein expression according to the manufacturer’s instructions. Transfection of H9c2 cells with siRNAs was carried out using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. H9c2 cells were treated with a mixture containing DMEM, lipo2000, and Nr4a1 siRNA for 4 h. Subsequently, the mixture was replaced with DMEM and cultured for 72 h. Specific siEGFR sequences were as follows: sense, UGGCCCAGAGUUCCCUGAAdTdT, and antisense, UUCAGGGAACUCUGGGCCAdTdT. Transfected cells were then treated with PA, as previously described.