Mirna precursor

At 24 h prior to transfection, cells were plated in growth medium without antibiotics. Transient transfections were conducted using miRNA precursors (Thermo Fisher Scientific, Waltham, MA, USA) by using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Control miRNA (miR-CON; AM17110)/miR-410-3p precursor (Catolog no. 4464084) was used for miRNA transfections followed by functional assays. All miRNA transfections were for 72 h. For PC3 and C42B cells, miR-410/scrambled miR-410 plasmid was stably transfected using lipofectamine 2000 followed by selection in 1 µg/mL of puromycin. pMIR-hsa-mir-410-GFP, miRNA vector (pMIR) with CMV promoter-driven expression of human pre-miRNA hsa-mir-410 and GFP reporter and the corresponding scrambled control was obtained from Vigene Biosciences (Rockville, MD, USA).

Primary mouse skeletal muscle cells were isolated as described^{43 (link)}. Hind limb muscles from 2-week-old C57BL/6J mice were removed and mechanically minced in PBS. Thereafter, enzymatic dissociation of satellite cells was performed with Collagenase and Dispase (Thermo Fisher Scientific, MA) and the suspension was passed through a 70 μm cell strainer. Differential plating was performed during the initial passages to enrich for myoblasts. Cells were grown on collagen-coated plates with growth media (DMEM/Ham’s F10 (1:1), 20% FBS, 1% penicillin-streptomycin/amphotericin B (anti-anti), and 2.5 ng mL⁻¹ bFGF). Differentiation was induced after plating 83,300 cells cm⁻² and ~12 h later switching to differentiation media (DMEM, 5% horse serum, and 1% anti-anti). Cultures were incubated at 37 °C in 5% CO₂ humidified chambers, and medium was changed every second day during growth and differentiation.
Cells were differentiated for 2 days and transfected with miRNA Pre-miR™ miRNA precursors (25 nM) for 5 h in OptiMEM reduced serum media with Lipofectamine® RNAiMAX. Two days after a single transfection, final experiments were performed. Media, miRNA precursors, and transfection reagents were from Thermo Fisher Scientific.

miRNA precursors, inhibitors, and negative controls were purchased from Ambion (Thermo Fisher Scientific, MA, USA). SIRT1 and Rab7 small interfering RNAs (siRNAs) were purchased from RiboBio (Guangzhou, China). Transfections with 50 nM miRNA and 100 nM siRNA were performed using Lipofectamine RNAiMAX (Invitrogen, NY, USA) according to the manufacturer's instructions.

Cells were plated in growth medium without antibiotics ~24 hrs before transfections. Transient transfections of miRNA precursor (Ambion) or siRNA (Origene) was carried out by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. For miRNA transfections, miR-3622b precursor (PM20243) or negative control (miR-CON) (AM17110) were purchased from Ambion. Trilencer-27 predesigned siRNA duplexes (SR301357) or universal scrambled negative control siRNA duplex (SR30004) purchased from Origene were used for siRNA-mediated EGFR knockdown. All miRNA/siRNA transfections were for 72h.