Ndufs2

Cells were fixed with 4% paraformaldehyde for 15 min and washed 3× with Tris-buffered saline (TBS) before the blocking solution consisting of TBS with 10% donkey serum, 0.1% Triton X-100 and 0.1% Tween-20 was applied for 1 h. All primary antibodies were diluted in blocking solution (MTCO2, Abcam; MTCO1, Abcam; NDUFS8, Abcam; NDUFS2, Abcam; GARS, Proteintech; S-100Beta, Abcam; Collagen 1, Abcam; Nestin, Milliopore; GRFS1, Abcam; eIF4E, Abcam; Mitotracker, Invitrogen). Incubation of the primary antibody was performed overnight at 4°C. The next day, cells were washed 3× in TBS before the secondary antibody (Alexa-Fluor Anti-Rabbit 488, Alexa-Fluor Anti-mouse 594, Invitrogen) and DAPI diluted in blocking solution was applied for 1.5 h at room temperature. Immunofluorescence images were collected using a Zeiss Axio Imager Z1 fluorescence microscope equipped with Zeiss Apotome 2 (Zeiss, Germany) in AxioVisionRel 4.9 software.

Total protein was harvested in RIPA lysis buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific, #A32959). Protein concentration was determined by Bradford assay (Bio-Rad), after which samples were subjected to SDS-PAGE and electroblotted onto Immobilon-P membrane (Millipore). Membranes were blocked in 5% nonfat milk or 5% bovine serum albumin (BSA) in TBST, before sequential probing with primary antibodies and HRP-conjugated secondary antibodies in blocking solution^{68 (link)}. Target proteins were visualized by ECL (Biorad) using ChemiDoc Imaging System (Biorad). Uncropped scans are shown in Supplementary Fig. 8. The following antibodies were used: OSMR (1:100, Santa Cruz, #271695), mtHSP70 (1:1000, Invitrogen, #MA3-028), TIM44 (1:500, Abcam, #244466), TOM20 (1:1000, Cell Signalling, #42406), H3K4me3 (1:1000, Abcam, #8580), BCL2 (1:1000, Cell Signalling, #15071), prohibitin (1:1000, Cell Signalling, #2426), NDUFS1 (1:3000, Abcam, #169540), NDUFS2 (1:4000, Abcam, #110249), α-tubulin (1:5000, Abcam, #4074), Na+/K+ ATPase (1:1000, Abcam, #58475), calnexin (1:1000, Abcam, #22595), phospho-Akt (Ser473) (1:1000, Cell Signalling, #4060), phospho-p44/42 MAPK (Thr202/Tyr204) (1:1000, Cell Signalling, #9101) and phospho-STAT3 (Tyr705) (1:1000, Cell Signalling, #9138).

AGK-Flag, NDUFS2-HA, and NDUFA10-HA plasmids were transfected into LO₂ cells using Lipofectamine 2000 (ThermoFisher, Waltham, MA, USA). The cells were stained with Flag antibodies (Sigma-Aldrich, Shanghai, China), HA antibodies (Proteintech, Rosemont, IL, USA), DAPI (Yeasen, Shanghai, China), and mitochondria-targeting dye (ThermoFisher), respectively. Hepatocytes were isolated from Agk^-/- and Agk^f/f mice, as described previously 19 (link). Cells were stained with AGK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NDUFS2 (Abcam, Cambridge, UK), NDUFA10 antibodies (Santa Cruz Biotechnology), and Lamp1 (Lysosomal-associated membrane protein 1) antibodies (Abcam), respectively. Immunofluorescence images were obtained using a laser scanning confocal microscope (ZEISS, Oberkochen, Germany) equipped with an Airyscan module on the Shanghai Jiao Tong University School of Medicine platform. LAS X 3.3.3 (Leica, Wetzlar, Germany) software was used for data acquisition and image processing.