Facl4

were Twinkle (mouse-monoclonal), kind gift from Prof. Anu Suomalainen-Wartiovaara (see also7 (link)); TFAM (rabbit-polyclonal), kind gift from Prof. Rudolf Wiesner; ATAD3 rabbit-polyclonal11 (link); coxII, Cyclophilin D, POLRMT, PHB1, HSP60 and Mitofilin, from Abcam; polG, coxII, TOM20, VDAC1 and FACL4, from Santa Cruz; MRPL49 and MRPS22, from Proteintech Europe; Calnexin from Cell signaling and SSB from Sigma Aldrich, Atlas.

Cells were grown on 14 mm round coverslips and fixed in 3% formaldehyde in PBS pH 7.4 containing 0.025% glutaraldehyde. Cells were permeabilized with 0.1% Triton and incubated with primary antibodies. Antibodies used were as follows: FACL4 (mouse monoclonal, 1:500, Santa Cruz Biotechnology Inc.); InsP₃R type 3 (mouse monoclonal, 1:500, Becton Dickinson Biosciences); rodent Nox4 (rabbit polyclonal, 1:500; Anilkumar et al, 2008). Secondary antibodies were as follows: goat anti‐mouse Alexa 568 and goat anti‐rabbit Alexa 488 (both 1:500, Thermo Fisher). Cells were mounted in Mowiol and imaging was performed on an inverted Nikon Ti‐E microscope equipped with a Yokogawa CSU‐X1 spinning‐disk confocal unit, an Andor DU‐897 camera, a Sutter filter wheel, and Nikon 100 × 1.49 NA objective (Nikon Instruments).