Mitotracker red fm

Open reading frames of candidate genes known or predicted to have a direct role in nuclear DNA repair were cloned into pENTR3C by InFusion Cloning (see Table S1 for all relevant primers). They were transferred from pENTR3C plasmids into the pAWG destination vector by Gateway Cloning (11791100; Thermo Fisher Scientific). S2R+ cells were transiently transfected using TransIT-Insect Transfection Reagent (MIR6100; Mirus Bio). One day after transfection, cells were incubated with 2 μM Hoechst (62249; Thermo Fisher Scientific) and 0.1 μM MitoTracker Red FM (8778; Cell Signaling Technology) and imaged on an SP5 Leica inverted confocal microscope.

To detect changes in mitochondrial morphology, cells were grown on glass coverslips, then transfected with negative control siRNA or CRIF1 siRNA for 48 h. After cells had been washed with PBS, they were stained with MitoTracker Red FM (Cell Signaling Technology, Topsfield, MA, USA) at 37 °C for 30 min in the dark. Subsequently, cells were washed with Hank’s balanced salt solution and mounted with Vectashield anti-fade mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI) (Vector Labs, Peterborough, UK). Images were obtained using a confocal laser microscope (LSM700, Carl Zeiss, Jena, Germany).