Mitochondrial isolation kit

Mitochondrial DNA (mtDNA) was isolated from iWAT and 3T3-L1 adipocytes using a Mitochondrial Isolation kit (Biovision, Milpitas, USA) and qPCR was performed to determine mtDNA copy number using mtDNA primers (Supplementary Information Table 1). Citrate synthase activity was measured in iWAT and 3T3-L1 adipocytes using a citrate synthase activity assay kit (Biovision, Milpitas, USA) according to the manufacturer’s instructions. For measurement of OCR, differentiated 3T3-L1 adipocytes were seeded onto XFp cell culture microplates (Seahorse Bioscience, North Billerica, USA) containing 80 μL of growth medium (Seahorse Bioscience, North Billerica, USA) and incubated in a CO₂ incubator at 37 °C for 24 h. A Seahorse Bioscience XFp analyzer was used to assess changes in dissolved oxygen levels in the media. All procedures followed the protocols recommended by the manufacturers.

Differential centrifugation was used to enrich the mitochondria in the mouse liver using a mitochondrial isolation kit purchased from Biovision (Milpitas Blvd, Milpitas, CA, USA) [36 (link)]. Briefly, after the livers were collected, washed, and homogenized using pre-cooled glass homogenizers, the resulting homogenates were immediately centrifuged at 600× g for 10 min with the isolation buffer provided by the kit. Subsequently, the supernatant was carefully collected and centrifuged at 7000× g for 10 min to precipitate the mitochondria, which were washed again by centrifugation with an isolation buffer. Finally, the supernatants were removed and the mitochondria were re-suspended in the storage buffer provided with the kit.

Mitochondria were isolated using the mitochondrial isolation kit (BioVision, Inc. CA, USA) according to the manufacturer’s protocol. In brief, after washing with ice-cold PBS, cells were centrifuged at 600g for 5 min and resuspended in 1× Cytosol Extraction Buffer. Then, homogenization was centrifuged at 1200g for 10 min at 4 °C to remove nuclei and intact cells. The collected supernatant was centrifuged at 10,000 g for 30 min at 4 °C. Finally, the pellets were resuspended in 1× Cytosol Extraction Buffer and centrifuged at 10,000g for 30 min at 4 °C to obtain mitochondria.

Mitochondria were isolated from the liver of wildtype C57BL/6 mice using a mitochondrial isolation kit (BioVision) following the manufacturer’s instructions. The nuclear fractions of hepatocytes were reserved for the subsequent preparation of nDNA.
MtDNA and nDNA were purified from mitochondrial pellets and nuclear fractions respectively using DNeasy blood and tissue kits (Qiagen, Hilden, Germany). Both mtDNA and nDNA were dissolved in sterilized 0.9% sodium chloride (NS). The DNA concentrations and purity were analyzed using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The quality of all DNA samples was checked on a NanoDrop2000 (Thermo Fisher Scientific, MA, USA). Furthermore, we confirmed that no detectable endotoxin levels in the samples (Associates of Cape Cod Inc., East Falmouth, MA, USA). To further exclude any significant nDNA contamination and to ensure the purity of the mtDNA, Q-PCR was conducted. The nDNA content was less than 0.1% in the isolated mtDNA samples.

Total cell lysates or mitochondrial lysates were used to determine NADPH levels in 3D culture conditions. Briefly, 20 × 10⁶ cells were cultured in ultralow attachment surface plates for at least 72 h before NADPH quantification. Mitochondrial Isolation Kit (BioVision) was used to obtain the mitochondrial lysates following the manufacturer’s instructions. NADPH levels were assessed in total cell lysates and mitochondrial lysates by using the NADP/NADPH Assay Kit (abcam) following the manufacturer’s instructions.

The extraction and purification of mitochondrial were administrated using a mitochondrial isolation kit (Biovision, USA) following the manufacturer’s instructions. In brief, after being washed several times and minced in ice-cold mitochondria isolation buffer, the gastric mucosal scrap from each group were collected and homogenized using Ultra-Turax T10 homogenizer (IKA, USA). The homogenate was transferred to a tube and centrifuged at 600×g for 10 min at 4°C. The supernatant was collected in a separate tube and centrifuge at 7,000×g for 10 min at 4°C. The supernatant was collected as cytosolic fraction. The mitochondria pellet was resuspended in storage buffer and identified with 0.5% Jenas Green B dye.