MitoSOX and MitoTRACKER fluorescence staining

Generation of superoxide in mitochondria was determined using MitoSOX Red fluorescent probe (Thermo Fisher Scientific), a lipophilic triphenylphosphonium cation containing dye that diffuses across the lipid membrane and selectively accumulates in mitochondria, where a red fluorescence is produced upon oxidation by superoxide [41]. To label mitochondria, the cell-permeant MitoTRACKER Green FM probe was used. MitoTRACKER contains a thiol reactive moiety and accumulates in the mitochondrial matrix covalently binding to mitochondrial proteins [42].