100 mm cell dishes

ASCs were transfected with miR-122 (Exiqon, Germatown, MD) per well mixed with the Lipofectamine RNAiMAX Reagent (Thermo). After 72hr of transfection, the cells were morphologically observed by the inverted microscope. The cell numbers of the experimental groups were counted automatic cell counter (Countess^®, Invitrogen, San Diego, CA, United States) using trypan blue solution. Transfected cells were processed for cell phenotyping or differentiated into three-lineage induction.
ASCs with or without miR-122 transfection were grown in a 100 mm cell dishes (Corning Glass Works, Corning, NY, United States). After reaching 70%-80% confluence, 1.0 × 10⁶ ASCs were cultured in 5 mL serum-free low-glucose DMEM for 48 h. Therefore, to obtain 0.2 mL amount of secretome from 1.0 × 10⁶ ASCs, the conditioned media were concentrated 25-fold using ultra filtration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3; Millipore, Bedford, MA, United States). We then injected 0.1 mL amount of secretome per mouse. This means that one mouse is injected with the secretome obtained from 5 × 10⁵ ASCs. In this study, NCM refers to the secretome shed from ASCs after 48 h of incubation, and MCM refers to the secretome shed from miR-122-transfected ASCs after 48 h of incubation.

ASCs were grown in 100-mm cell dishes (Corning Glass Works). After reaching 70%–80% confluence, ASCs were transiently transfected with 4-µg pcDNA-PGC-1α. Twenty-four hours later, 1.0 × 10⁶ ASCs were cultured in 7-mL serum-free low-glucose DMEM for 24 hours. The conditioned media were concentrated 25-fold using ultrafiltration units with a 3-kDa molecular weight cutoff (Amicon Ultra-PL 3) to obtain 0.28 mL of secretome from 1.0 × 10⁶ ASCs. Mice were injected with 0.1 mL of secretome and PGC-1α–boosted secretome (PGC-Sec), equivalent to the secretome obtained from 5 × 10⁵ ASCs. The normal secretome was obtained from empty vector-transfected ASCs, while the PGC-Sec was obtained from pcDNA-PGC-1α–transfected ASCs.