Afg3l2

Human primary liver cancer cell lines including HCC cells MHCC-97 H, Huh7, and ICC cells QBC939, CCLP1 were all purchased from Cell Bank of the Typical Culture Preservation Committee, Chinese Academy of Sciences (Shanghai, China). MHCC-97 H, Huh7, QBC939 and CCLP1 cells were cultured in the DMEM medium (Invitrogen, Shanghai, China). All mediums were supplemented with 10% FBS and 100U/mL penicillin–streptomycin. The following commercially available antibodies were used for Western blotting: GAPDH (Abcam, ab75834), FDX1 (Abcam, ab108257), Actin (Abclonal, ac026), DLAT (CST, 12,362S), LIAS (Proteintech, 11577-1-AP), Lipoic acid antibody (Abcam, ab58724), AFG3L2 (Proteintech, 14631-1-AP), ATF4 (CST, 11,815), p62 (MBL, PM045). The chemical reagents used in this study included L-Glutathione (reduced) (MCE, HY-D0187), Sorafenib(Selleck, S7397), Erastin(MCE, HY-15,763), Ammonium tetrathiomolybdate (TTM) (Macklin, A828261), Elesclomol (MCE, HY-12,040), Ditiocarb sodium (MCE, HY-B1637), Baf-A1 (Sigma Aldrich, B1793), cycloheximide (Sigma Aldrich, C7698), MG-132 (Sigma Aldrich, 474,790), MitoTracker Red CMXRos (Thermo Fisher Scientific, M7512), Z-VAD-FMK (MCE, HY-16658B), BOC-D-FMK (MCE, HY-13,229), Tetraethylenepentamine (TEPA) (Macklin, T818822), BODIPY™ 581/591 C11 (Invitrogen, D3861).

Western blotting was performed as previously described^{46 (link)}. Electron transport proteins were detected using a total OXPHOS human western blot antibody cocktail (ab110411) from Abcam. The primary antibodies used are SFXN4 (Thermofischer (PA5-35980)), P-Histone H2A.X (Cell Signaling Technology (9718S)), Histone H2A.X (Cell Signaling Technology (2595S)), XPD (D3Z6I) (Cell Signaling Technology (11963)), NTH1 (Abcam (ab236912)), MUTYH (Abcam (ab228722)), FANCJ (BACH1/BRIP1) (Cell Signaling Technology (4578)), POLD1 (BD Biosciences (610972)), FTH^{46 (link)}, TFR1 (Thermo Fisher (13–6800)), HSP60 (Cell Signaling Technology (12165S)), AFG3L2 (Proteintech (14631-1-AP)), HSP90 (Cell Signaling Technology (4877), Tubulin (Cell Signaling Technology (12351)), β-Actin (Sigma Aldrich (A3854) C/EBPβ Antibody (Cell Signaling Technology #9008),Id2 (D39E8) (Cell Signaling Technology #3431) and RPA32/RPA2 Antibody (Cell Signaling Technology #52448).

Cells were solubilized in phosphate buffered saline, 1% dodecyl-maltoside (DDM), 1 mm phenylmethylsulfonyl fluoride (PMSF) and complete protease inhibitor (Thermo Fisher). Protein concentrations were measured by the Bradford assay (BioRad). Equal amounts of proteins were separated by Tris-Glycine SDS-PAGE and were transferred to nitrocellulose membrane by semi-dry transfer. Membranes were blocked in TBST with 1% milk at room temperature for 1 h, followed by incubation with primary antibodies overnight at 4°C in 5% BSA/TBST. Signals were detected the following day with secondary HRP conjugates (Jackson ImmunoResearch) using ECL with X-ray film and iBright Imaging System (Thermo Fisher). Primary antibodies from Proteintech Group: AFG3L2 (14631-1-AP, 1:5000), uL11m (15543-1-AP, 1:20 000), mS27 (17280-1-AP, 1:5000) and OXA1L (21055-1-AP, 1:5000). Additional primary antibodies used were MTCO1(1D6E1A8, 1:500) (Abcam/Mitosciences) and TOM40 (sc-11 414, 1:5000) (Santa Cruz). Representative data of independent experiments were cropped in Adobe Photoshop with only linear corrections to brightness applied.