Phox i2

For live cell analyses cells were grown on collagen-coated (0.1 mg/ml) glass slides and mitochondrial morphology was analyzed by CMXRos-staining (300 nM). Images were collected using a 63x oil objective of an Axiovert200M microscope equipped with an ApoTome.2 system for structured illumination microscopy (Zeiss, Vienna, Austria)^{17 (link),21 (link)}. Images were taken at equal exposure times and processed using Axiovision software. For quantification, at least 100 cells from three independent experiments were analyzed for their mitochondrial morphology/number of branches. Mitochondria with more than 50 branches per 30 × 30 µm were classified as “small tubular”, mitochondria with more than 5 slubs/dots per 30 × 30 µm were classified as fusion defect. For ROS-measurements, the cells were grown on 8 µm chamber slides (ibidi, Martinsried, Germany) and were incubated with CM-H₂XROS (final concentration 500 nM; Invitrogen, USA). Images were acquired with a Zeiss Axiovert200M microscope and cellular fluorescence intensity was quantified using Axiovision software (Zeiss, Vienna, Austria). Phox-I2 was purchased from Sigma-Aldrich (Vienna, Austria) and added 24 hours before microscopy.

RPMI 1640 with Glutamax, DMEM/F12 (1:1), Hanks' buffered salt solution (HBSS), fetal bovine serum (FBS), and Amplex red were purchased from Invitrogen, Paisley, UK. Pest (penicillin, streptomycin), neomycin, ionomycin, phorbolmyristateacetate (PMA), diphenyleneiodoniumchloride (DPI), dapsone, ML-171, Phox-I2, xanthine, hypoxanthine, xanthine oxidase, DMSO, DPPH (2,2-diphenyl-1-1picrylhydrazyl), Tween20, Sucrose, flavin adenine dinucleotide (FAD), Phosphatidic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), horseradish peroxidase (HRP) and NADPH were purchased from Sigma–Aldrich. HEK293 overexpressing Nox4 (CJ Nox4) cells were purchased from Redoxis, Lund, Sweden. HEK 293 cells expressing Nox5 and CHO cells expressing Nox1 were a kind gift from the Vincent Jaque Center Medical Universitaire, Geneva, Switzerland. GKT136901, a Nox1/Nox4 selective inhibitor, was a kind gift from prof. Harald HH Schmidt (Maastricht University, Netherlands).