Control fibroblasts were transfected with siRNA directed against WFS1 (ON-TARGETplus Human WFS1 siRNA SMARTpool, Thermo Fisher Scientific Biosciences, Waltham, MA, USA) and/or with siRNA directed against NCS1 (ON-TARGETplus Human NCS1 siRNA SMARTpool, Thermo Fisher Scientific Biosciences) using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions and processed 72 or 96 hours later for Western blot or oxygraphy analyses. Transfection with ON-TARGET Non-Targeting Pool (siScr) was used as controls.

To overexpress NCS1-Flag or pericam-mt vectors, fibroblasts were transfected by nucleofection. Nucleofection was performed according to an optimized protocol provided by the manufacturer (Amaxa Biosystems, Cologne, Germany). Cells were suspended in 100 μl of nucleofactor solution (Amaxa Biosystems); mixed with pericam-mt, Flag, or NCS1-Flag construct; and subjected to nucleofection, after which cells were transferred into prewarmed fresh medium. Cells were analyzed 24 hours after nucleofection.

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