Succinate assay kit

The AcAc content in LO2 cells was measured by human acetoacetate ELISA Kit (Shanghai J&L Biological, China). The succinate and fumarate content of LO2 cells were measured using the Succinate Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA) and the Fumarate Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s instructions.

ADSCs (2 × 10⁶ in quantity) transfected with shNC or shBcat1#1 were used for intracellular metabolite concentration measurement. Each metabolite level was determined using the following assay kits: BCAA detection kit (BioVision, K564‐100, α‐ketoglutarate assay kit (Sigma‐Aldrich, MAK054), glutamate assay kit (Sigma‐Aldrich, MAK004), citrate assay kit (Sigma‐Aldrich, MAK057), fumarate assay kit (Sigma‐Aldrich, MAK060), succinate assay kit (Sigma‐Aldrich, MAK184), and malate assay kit (Sigma‐Aldrich, MAK196). BCKA concentration was measured by high‐performance liquid chromatography (HPLC) as previously described.^[42] Intracellular metabolite levels were normalized to the ADSCs transfected with shNC.

Glucose levels were determined in conditioned media of H1993 and H1975 cells that were treated or not with KU55933. Briefly, conditioned media were removed, centrifuged at 13,000 g at 4 °C for 10 min, and then assayed for glucose concentrations using the Glucose Assay Kit (Sigma-Aldrich), following manufacturer’s instructions. Moreover, intracellular citrate, pyruvate, and succinate levels were determined in H1993 and H1975 cells using the Citrate Assay Kit (Sigma-Aldrich), Pyruvate Assay Kit (Sigma-Aldrich), and Succinate Assay Kit (Sigma-Aldrich) following manufacturers’ instructions. Briefly, cells were seeded in six-well flat-bottomed plates at a density of 3 × 10⁵ cells per well and then treated with ATM inhibitor (100-nM) for 48 h. Cells were collected and resuspended in specific substrate assay buffer and incubated with appropriate assay mix. The optical density (OD) was measured using microplate spectrophotometer, and metabolite concentrations were calculated from the corresponding standard curve and normalized to 10⁶ cells. At least three independent experiments were performed, and data were pooled.