Sirnas

Scrambled siRNA (5′- GGC CGA UUG UCA AAU AAU U- 3′), siRNAs against FDXR (5′-CAC CUU GAU CCA GCG GAC UUA -3′ and 5′- GCU CAG CAG CAU UGG GUA U -3′), siRNAs against FDX2 (5′- GCU GCA AUA AAU CGA UAA CAC -3′ and 5′- GCU GCC AGA UUG UCU GAC AC -3′) and siRNA against IRP2 (5′- GCA AAC AUG UGU CCG GAA U -3′) were purchased from Dharmacon (Chicago, IL, USA). RNAiMax from Life Technology (Carlsbad, CA, USA) was used for siRNA transfection according to the user manual. Cells were transfected with scrambled or target siRNA at 25 nM for 3 days.

Single myofibers were isolated from EDL muscles of 6–8 week old mice as previously described (Pasut et al., 2013 (link)). Briefly, EDL muscles were dissected and incubated in DMEM (Gibco) containing 0.2% collagenase I (Worthington) for 1 hour. Myofibers were detached using gentle trituration with a glass pipette and washed several times with DMEM. Myofibers were fixed with 2% PFA for 10 min either immediately after isolation, after 36 hours (to analyze when satellite cells are entering the cell cycle), or 42 hours (to analyze when satellite cells have undergone their first division) in culture. For culturing, myofibers were maintained in DMEM high glucose (Life Technologies) supplemented with 20% fetal bovine serum, 1% chick embryo extract and 2.5 ng/ml bFGF. siRNA transfections were performed at 4h and 16h post-culture using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. siRNAs were purchased from Dharmacon (Table S3) and used at a final concentration of 50 nM. For the assessment of symmetric and asymmetric satellite stem cell divisions, EDL myofibers were isolated from 9 Myf5-Cre:R26R-EYFP mice. Fibers from each mouse were subsequently treated with siRNA, and approximately 300 satellite cells from 30–35 myofibers were analyzed per condition.