Knockdown of Pfkfb3 and Pdk4 in cultured muscle fibers

One hundred of the fibers prepared above were cultured on a plate coated with Matrigel matrix (Corning Incorporated, Corning, NY, USA) with 500 μl standard medium (DMEM containing 25 mM glucose supplemented with 20% fetal bovine serum (Thermo Fisher Scientific), 2% chick embryo extract (United States Biological, Salem, MA, USA), and 1% penicillin–streptomycin (PS, 10,000 U/ml, Thermo Fisher Scientific) at 37 °C under 5% CO₂ for 24 h. A mixture of two Pfkfb3 siRNA duplexes (siPfkfb3, 200 nM, SASI_Mm01_00034119 and SASI_Mm01_00034121 (Sigma-Aldrich)) or two Pdk4 siRNA duplexes (siPdk4, 100 nM, SASI_Mm01_00053023 and SASI_Mm01_00053024 (Sigma-Aldrich)) were transfected into the fibers using Lipofectamine RNAiMAX (Thermo Fisher Scientific) for 6, 9, 12, or 24 h in standard medium without PS. Stealth RNAi^TM siRNA Negative Control, Med GC (Thermo Fisher Scientific) was used as a negative control. Transfection was performed according to the manufacturer’s protocol and the procedure described by Huttner et al. ⁸². RNAs were prepared from the fibers and then subjected to qRT-PCR of Pfkfb3 and Pdk4.