Mmir3427 mr04

Prior to transfection, MSCs were dissociated using TrypLE Express, washed twice with PBS, and placed in 100 μL of Opti‐MEM (Thermo Fisher Scientific). MSCs were then transfected with miExpress™ EGFP‐mmu‐miR‐155 plasmid (GeneCopoeia Inc. Rockville, MD, USA) or pPBQM‐mmu‐miR‐155 plasmid (a gift from Dr. Martin Lotz) using a CUY21 electroporator (NEPA Gene, Tokyo, Japan). In the miExpress system, the precursor miRNA is placed in the 3′UTR region of the EGFP gene. After transfection, the precursors were co‐expressed with EGFP and processed in the cells. We used a scrambled control sequence expression plasmid (CmiR0001‐MR04; GeneCopoeia, Inc.) and a mmu‐miR‐155‐5p precursor expression plasmid (MmiR3427‐MR04, GeneCopoeia, Inc.). The pPBQM‐miR‐155 plasmid consists of the precursor sequence of mmu‐miR‐155 controlled by a cumate‐gene switch (Mullick et al., 2006), an EF1‐CymR repressor cassette, and a piggyback transposon backbone. The cumate‐gene switch was activated by adding 30 μg mL⁻¹ cumate (QM100A‐1; System Bioscience Inc., Palo Alto, CA, USA). The C/ebpβ expression plasmid was from Addgene (#12557, Cambridge, MA, USA).
For mimic miRNA transfection experiments in human MSCs, we used a miRNA mimic for hsa‐miR‐155‐5p (mirVana^® miRNA mimic, MC12601, Thermo Fisher Scientific) with Lipofectamine^® RNAiMAX (Thermo Fisher Scientific) following the manufacturer's instructions.

The mouse myoblast cell line C2C12 was cultured in DMEM (Wako, Tokyo, Japan) supplemented with 200 mM L-glutamine and 10% fetal bovine serum (Hyclone, Logan, UT, USA), in 5% CO₂ at 37°C. For overexpression of miR-155, C2C12 cells at 80% confluency were transfected with miExpress EGFP-mmu-miR-155 plasmid (GeneCopoeia Inc. Rockville, MD, USA) or pPBQM-mmu-miR-155 plasmid (a gift from Dr. Martin Lotz) using ScreenFect A (Wako). We also used a scrambled control sequence expression plasmid (CmiR0001-MR04, GeneCopoeia, Inc.) and a mmu-miR-155 precursor expression plasmid (MmiR3427-MR04, GeneCopoeia, Inc.). The cumate-gene switch was activated by adding 30 μg/mL cumate (QM100A-1, System Bioscience Inc., Palo Alto, CA, USA). Myogenic differentiation was induced by culturing confluent C2C12 cells in DMEM containing 2% horse serum (Biowest USA, NW, USA) for 12 days.