Mitotracker deep red dye

Cells (10⁵) were incubated with 200 nM MitoTracker Deep Red dye (Thermo-Fischer Scientific) for 30 min, and transferred to 0.01% poly-L-lysine (Sigma-Aldrich) coated glass slides (Thermo-Fisher Scientific). Next, cells were fixed 15 min in 4% formaldehyde then cold 100% methanol and mounted in ProLongTM Gold antifade medium with DAPI (Invitrogen). Images were acquired using a Zeiss LSM 800 microscope with Airyscan.

Mitochondria were visualized with MitoTracker DeepRed dye (Thermo Fisher Scientific, Waltham, Massachusetts, USA), which stains mitochondria in living cells regardless of mitochondrial membrane potential. The dye solution was added to wells, and plates were incubated for 10 minutes at 37°C. The confocal images were collected with the Olympus IX70 FV500 confocal microscope equipped with an oil 60x UPlanApo lens. A He-Ne 633 nm laser used as a light source, and the fluorescence signal was collected through the 660 nm BA filter.

In order to visualize mitochondrial DNA nucleoids, fibroblasts were stained with PicoGreen (Thermo Fisher Scientific, P7581) as described previously [7 (link)]. Briefly, cells were seeded on glass bottom dishes (Mattek, P35G-1.5-14-C) at 8 × 10⁴ and incubated overnight. Approximately 1 h prior to imaging, cells were stained with PicoGreen at 3 µL/mL for 30–45 min at 37 °C. MitoTracker Red dye (50 nM) (Thermo Fisher Scientific, M7512) or MitoTracker Deep Red dye (50 nM) (ThermoFischer Scientific, M22426) was also added to the media to visualize mitochondrial networks. The media containing dyes was aspirated, cells were washed four times in pre-warmed 1XPBS, and fresh pre-warmed media was added to the cells.

Met was purchased from Solarbio (Beijing, China). Staurosporine (STS), Caspase-3/8/9 Assay Kit, ROS Assay Kit and JC-1 Assay Kit were provided by Beyotime (Shanghai, China). Anti-PARP (9532), anti-Bak (12105), anti-Bax (5023), anti-Bcl-xL (2764), anti-Mcl-1 (94296), anti-Bim (2933), anti-Bad (9268), anti-Puma (98672), anti-AMPK (5831), anti-p-AMPK (8208), anti-Cleaved Caspase-3 (9664), anti-Cleaved Caspase-8 (8592), anti-Caspase-9 (9502) Rabbit monoclonal antibody, and anti-Bid (8762) Mouse monoclonal antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH (sc-47724) Mouse monoclonal antibody was obtained from Santa Cruz (Texas, USA). Luciferase Mycoplasma Detection Kit and anti-β-tubulin (HC101) Mouse monoclonal antibody were purchased from Transgen Biotech (Beijing, China). Turbofect™ transfection regent and Mito-tracker Deep Red dye were obtained from ThermoFisher Scientific (Massachusetts, USA). Cell counting Kit-8 (CCK-8) and Alexa Fluor^® 488 annexin V/Dead Cell Apoptosis Kit were purchased from Dojindo (Kyushu, Japan). Acetylcysteien (NAC), Z-VAD-(OMe)-FMK (Z-VAD) and Compound C were purchased from MedChemExpress (New Jersey, USA). Hoechst 33258 dye and PI dye were obtained from G-clone (Beijing, China).