HeLa cells were fixed for 20 min with PBS containing 3.7% formaldehyde, permeabilized for 5 min in 1× PBS, 0.25% Triton X-100 and incubated for 10 min at −20°C with methanol prior to nuclear NRF2 detection. Non-specific binding sites were blocked with 3% bovine serum albumin in 1× PBS for 15-30 min at room temperature. Cells were immunostained with rabbit polyclonal anti-NRF2 antibody (1:50; sc-13032 Santa Cruz Biotechnology) for 1 h at 37°C. HeLa cells were then incubated with Alexa Fluor 488-conjugated secondary antibodies (1:300; Molecular Probes), labelled with 0.25 µg/ml Hoechst in 1× PBS for 5 min and mounted in Mowiol. Immunolabelling of HeLa cells 66, 67, 68, 69, 70 and 72 h after siCtrl or siOPA1 transfection was visualized under a fluorescence microscope (Nikon Eclipse 80i), and the images were acquired using an NIS-Element (Nikon Digital Sight DUS2 camera). HeLa cells showing accumulation of NRF2 staining in the nucleus were counted by stack with Hoechst labelling of the nucleus using ImageJ software.
The mitochondrial network was stained with MitoTracker Red (M22425, Invitrogen) in culture medium for 15 min at 37°C and 5% CO2. Then, cells were fixed for 20 min with PBS containing 3.7% formaldehyde, permeabilized for 5 min in 1× PBS, 0.25% Triton™ X-100 and mounted in DAPI-mounting medium (VECTASHIELD With DAPI, H-1200-10, Eurobio Scientific).
The mitochondrial network was stained with MitoTracker Red (M22425, Invitrogen) in culture medium for 15 min at 37°C and 5% CO2. Then, cells were fixed for 20 min with PBS containing 3.7% formaldehyde, permeabilized for 5 min in 1× PBS, 0.25% Triton™ X-100 and mounted in DAPI-mounting medium (VECTASHIELD With DAPI, H-1200-10, Eurobio Scientific).