Mtco2

BCA was purchased from Selleck Chemicals (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Kumamoto, Japan). PE Annexin V apoptosis detection commercial kit was purchased from BD biosciences (Shanghai, China). Reactive Oxygen Species (ROS) Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China). Cell-Light EdU Apollo567 In Vitro Kit was purchased from Guangzhou Ruibo Biotechnology Co. LTD (Guangzhou, China). Bax, Bcl-2, ND1, SHDB, UQCRC2, MTCO2, ATP5A, MFN2, GLUT1, HK2, PMK2, LDH, HIF-1a, and Drp1 antibodies were obtained from proteintech (Wuhan, China). AKT, p-AKT, mTOR, and p-mTOR antibodies were obtained from Cell Signaling Technology. LC3B, Beclin-1, P62, and GAPDH were obtained from ABCAm (Cambridge, UK).

The protein was extracted from cultured NSC34-G93A cells using Laemmli SDS loading buffer and heated for 8 min at 90 °C. Protein samples were resolved via SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a PVDF membrane with a Bio-Rad semidry transfer cell (1703940, Plano, TX, USA). Membranes were blocked with 5% milk in TBS with 0.1% Tween-20 for 1 h. The primary antibodies used in this study were PGC1α (Abcam ab110324), MT-CO1 (CST 62101S), MT-CO2 (Proteintech 55070-1-AP), COX4 (CST 4844S), and Actin (CST 4970S). After overnight incubation with primary antibody at 4 °C, the membranes were washed with TBST buffer and then incubated with horseradish-peroxidase-conjugated secondary antibody at room temperate for 2 h. Protein bands were visualized using a Bio-Rad Clarity ECL kit under the ChemiDoc Imaging System (Bio-Rad Laboratory). The band intensity was analyzed using ImageJ software.

Tissues were lysed on ice in lysis-buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 6 mM EGTA, 20 mM NaF, 1% Triton X-100, and protease inhibitors) for 15–20 min. After centrifugation at 13000 rpm for 15 min, supernatants were collected for protein determinations and SDS-PAGE analysis. The following antibodies were used: pS245-Hdac4 (#3443), Hdac4 (#7628), Sik2 (#6919), Cox4 (4850), Pparg (#2435), and phosphor-PKA substrate (#9624) antibodies (Cell Signaling Technology), Hsp90 (Santa Cruz Biotechnology, #SC-7947), Ucp1 (Sigma, U6382), mt-Co1 (Abcam, #ab110413), mt-Co2 (Proteintech, #55070–1-AP), Cox5b (Bethyl, #A-305–523A), Cox6b (Abgent, #AP20624a), Hsp60 (Bethyl, #A302–846A), Hk1 (Proteintch, #19662-1-AP). Immunoblots were quantified using Image J software.