Cox 4

Approximately 1 × 10⁵ MEFs were plated in chambers and cultured at 37 °C in a 5% CO₂ incubator for 48 h. Then, cells were washed twice with PBS. Fixed cells with 4% formaldehyde, 95% ethanol, 2.5% glutaraldehyde, or the mixture of 3% formaldehyde and 1.5% glutaraldehyde for 20 min at room temperature, respectively. The coverslips were heated in antigen retrieval buffer (100 mM Tris, 5% (w/v) urea, pH 9.5) at 95 °C for 10 min. Following fixation, cells were washed 3 times in PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were washed twice for 5 min and blocked with 8% goat serum in PBS for 1 h. Primary antibodies VADC1 (5 μg/mL) and COX IV (10 μg/mL) (Abcam) were incubated overnight at 4 °C in 2% goat serum in PBS after washed once in PBS for 5 min. Then cells were washed in 1% goat serum, 0.1% Tween-20 in PBS 4 times for 10 min. Second antibodies (2 μg/mL) (Invitrogen) were incubated overnight at 4 °C in 2% goat serum in PBS for 1 h after washed in 1% goat serum, 0.1% Tween-20 (Sigma) in PBS 4 times for 10 min. Lastly, added a small amount of ProLong Gold antifade reagent with DAPI (Invitrogen) over the area where each chamber used to be. Cell imaging used a Zeiss LSM 800 confocal microscope (Zeiss, Oberkochen, Germany) equipped with a 60× 1.3 NA oil immersion objective.

The following antibodies and reagents were used: pα-Syn (Ser129, Biolegend, 825701), pα-Syn (Ser129, Cell Signaling Technology, 23706s), MAP2 (Thermo Fisher Scientific, SF254293), COX IV (Abcam, ab16056), ATG5 (Proteintech, 10181-2-AP), Beclin1 (Proteintech, 11306-1-AP), LC3 (Cell Signaling Technology, 12741), Bcl2 (Cell Signaling Technology, 3498S), Bax (Proteintech, 50599-2-Ig), GAPDH (Proteintech, 60004-1-Ig), TH (Sigma-Aldrich, AB152), Ubiquitin (Santa Cruz Biotechnology, sc-8017), Iba-1 (Wako, 019-19741), Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen, A-11005), Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, A-11012), DAPI (Biofroxx, EZ3412B205), HRP-conjugated anti-mouse IgG (BIO-RAD, 170-6516), HRP-conjugated anti-rabbit IgG (BIO-RAD, 170-6515), Complex I Enzyme Activity Microplate Assay Kit (Abcam, ab109721), and Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1).

Anti-p-PDHE1α (S293, S300) (Calbiochem, San Diego, CA, USA, AP1062-1064, respectively) were used for immunohistochemistry. Anti-p-PDHE1α (S293, S300) (Abfrontier), Anti-SMAD1/5/8 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-6031-R), anti-p-SMAD 1/5/8 (Cell Signaling Technology, Danvers, MA, USA, #9511), COXIV (Abcam, Cambridge, UK, ab16056) and anti-Flag (Sigma-Aldrich, F1804) antibodies were used for both immunofluorescence and western blotting. Anti-SMAD1 (Invitrogen, Carlsbad, CA, USA, #38-5400), anti-SMAD5 (Abgent, San Diego, CA, USA, AJ1726a), and Anti-SMAD1/5 (Santa Cruz Biotechnology, sc-6201) were used for immunofluorescence. Anti-α-tubulin (Applied Biological Materials, G098), anti-lamin B (Santa Cruz Biotechnology, sc-6216), anti-SMAD4 (Cell Signaling Technology, #9515) and anti-PDK4 serum (Abfrontier) were used for western blotting.

Antibodies against Drp1, p62, LC3, RIP1, RIP3, TSG101, and CD63 were purchased from Abcam (Cambridge, MA, USA). β-actin, tubulin, and COX IV, which were used as references for the total, cytoplasmic, and mitochondrial fractions, were also acquired from Abcam (Cambridge, MA, USA). Antibodies against Phospho-RIP3 (Thr231/Ser232) and Phospho-Drp1 (Ser616) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Aggresome Detection Kit was acquired from Abcam (Cambridge, MA, USA). MitoTracker Deep Red, the ROS 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit, dihydroethidium (DHE) assay kit, and MitoSOX detection kit were purchased from Invitrogen (Carlsbad, CA, USA). Ad-mRFP-GFP-LC3 was supplied by Beyotime (Shanghai, China). Adenoviral vectors for Drp1 short hairpin RNA (shRNA) and Drp1 mutations (Drp1-S616D and Drp1-S616A) were generated by Genechem Technology (Shanghai, China). Related activators and inhibitors, including Mitochondrial division inhibitor 1 (Mdivi-1), N-acetylcysteine (NAC), and Necrostatin-1 (Nec-1), were obtained from Selleck (Shanghai, China). The Protein A/G Magnetic Beads IP Kit was purchased from Thermo Scientific (Waltham, MA, USA), and fetal bovine serum (FBS) and penicillin/streptomycin were procured by Invitrogen (Carlsbad, CA, USA). All other chemicals were supplied by Sigma unless otherwise specified.