Transfection of small interfering RNAs (siRNAs) and overexpression plasmids
siRNA transfection was performed using Lipofectamine RNAiMAX transfection reagent (Life Technology, New York, NY) by following the manufacturer’s guidelines. Briefly, C2C12 cells were seeded into a six-well plate. After four days of differentiation, 5 pmol of siRNA was diluted with Opti-MEM and mixed with the transfection reagent, and the mixture was added to each well. After transfection for 24 h, the cells were challenged with different treatments; some were co-cultured with C26 cell-conditioned media (C26 CM), and some were cultured in DMEM. The siRNAs specific to HSP90aa1 were purchased from Thermo (#159050,Life Technology, New York, NY). The siRNAs specific to FOXO1 were purchased from Santa Cruz (#56458,Santa Cruz, TX).
The constitutively activated STAT3 plasmids (STAT3-C Flag pRc/CMV) were purchased from Addgene (Plasmid #8722). C2C12 cells were transiently transfected with STAT3-C plasmids using the Lipofectamine™ 2000 Transfection Reagent according to the manufacturer’s instructions (Life Technology, New York, NY).