Fibroblasts were generated from a 6‐mm skin punch biopsy taken under local anesthetic of healthy control individuals (Ctrl1: 35‐year‐old male, Ctrl2: 25‐year‐old female, Ctrl3: 57‐year‐old male) and patients carrying a c.22 G>A, p.Val8Met mutation in MSTO1 gene (MSTO P1, MSTO P2). Cells were grown in Dulbecco's modified Eagle's medium (DMEM), with 4.5 g/l glucose supplemented with 10% fetal bovine serum, 2 mM glutamate, 100 units/ml penicillin/streptomycin (all from GIBCO, Life Technology), and 110 mg/l pyruvate (Sigma). All cells were grown at 37°C in a humidified 5% CO2 incubator. HeLa cells (ATCC) were grown in the above‐described medium without pyruvate.
Before silencing, the cells were plated in antibiotic‐free medium, and the samples were silenced in serum‐free culture medium with MSTO1‐specific (s30301, Life Technologies) or scrambled siRNAs (Silencer® Negative Control #1 siRNA, Life Technologies) (100 nM), using Oligofectamine (Life Technologies) for 72 h. The efficacy of silencing was verified with Western blotting. For evaluation of mitochondrial morphology and fusion dynamics, the cells were transfected with mtDsRed1 or mtDsRed1‐T2A‐MSTO1 and mtPA‐GFP plasmid DNAs 24 h before the experiment.
Before silencing, the cells were plated in antibiotic‐free medium, and the samples were silenced in serum‐free culture medium with MSTO1‐specific (s30301, Life Technologies) or scrambled siRNAs (Silencer® Negative Control #1 siRNA, Life Technologies) (100 nM), using Oligofectamine (Life Technologies) for 72 h. The efficacy of silencing was verified with Western blotting. For evaluation of mitochondrial morphology and fusion dynamics, the cells were transfected with mtDsRed1 or mtDsRed1‐T2A‐MSTO1 and mtPA‐GFP plasmid DNAs 24 h before the experiment.