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).