Tomm20 f 10

Cells were lysed in buffer containing 0.5% NP-40 (Roche, 11754599001), 100 mM Tris-HCl (pH 7.4), and 300 mM NaCl and complemented with protease and phosphatase inhibitors [19 (link)]. Proteins were loaded by SDS-gel electrophoresis, and blots were probed with antibodies against glyceraldehyde 3-phosphate dehydrogenase(GAPDH) (6C5) (Millipore, NG1740950, dilution 1:5000), MLKL (D2I6N) (N° 14993, dilution 1:2500), p-MLKL (Ser358)(D6H3V) (N° 91689, dilution 1:2500), RIP1 (D94C12, dilution 1:1000) (N° 3493), prohibitin-1 (PHB1) (N° 2426S, dilution 1:1000) and p-RIP1 (Ser166)(D1L3S) (N° 65746, dilution 1:1000) from Cell Signaling, Lon peptidase 1 (LONP1) (Proteintech, 15440-1-AP, dilution 1:1000), p-RIP3 (Ser227) (EPR9627) (Abcam, ab209384, dilution 1:8000), and translocase of outer mitochondrial membrane 20 (TOMM20) (F-10) (Santa-Cruz, sc-17764, dilution 1:1000). Western blot images were analyzed by ImageJ software.

Cells were plated on pretreated coverslips with poly-L-lysine. After rotenone treatment, cells were washed with annexin buffer 1X and incubated with annexin V-FITC (1X) for 15 min at room temperature (RT). Subsequently, 5 μL of PI was directly added to cells. Thereafter, cells were washed and incubated with annexin buffer 1X to wipe away excess dye. Annexin/PI staining was observed by in vivo immunofluorescence. Nuclei were stained with 300 nM of 4′, 6-diamidino-2-phenylindole (DAPI) (Thermo Fisher, D1306). For mitochondrial morphology, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T9284). Once permeabilized, cells were incubated with bovine serum albumin (BSA)/PBS solution (1 mg/mL) for 1 h. Thereafter, cells were incubated for 1 h with TOMM20 (F10) (Santa-Cruz, sc-17764, dilution 1:200) and, subsequently, with Thermo Fisher Alexa Fluor^® 568 (A11004)-conjugated secondary antibodies for another hour at RT. Results were analyzed, and represented the proportion of cells with filamentous and damaged mitochondria for each condition. Images were visualized using an Olympus IX51 inverted microscope equipped with a DP71 camera.