Ab83431

The production of ATP (Cat No: BC0300, Solarbio, China), LA (Cat No: BC2235, Solarbio, China), glucose (Cat No: BC2505, Solarbio, China), and α-KG (Cat No: ab83431, Abcam, USA) were conducted according to the manufacturer's instructions.

2-KG was quantified by an ELISA kit from Abcam (ab83431) according to the manufacturers’ instructions.

α‐Ketoglutarate levels were measured using an α‐ketoglutarate assay kit (ab83431, Abcam, UK). Samples preparation and the α‐ketoglutarate assay were conducted according to the manufacturer's instructions. Lysates of HeLa cells were incubated with an enzyme reaction mixture for 30 min at 37 °C for transamination of α‐ketoglutarate, after which the generated pyruvate reacted with a probe that generated a specific color. The samples were then measured using a microplate reader (Synergy H1 Hybrid Reader, BioTek, VT, USA) at 570 nm. Finally, the concentration of α‐ketoglutarate was calculated using a standard curve.

According to the kit instructions (Abcam, ab83431), MDA-MB-231 cells (shcontrol or shP4HA1) and MCF10A cells (control or P4HA1, P4HB expressing) were washed with cold PBS and resuspended in 500 μl ice-cold α-KG Assay Buffer on ice. Cells were homogenized quickly by pipetting up and down a few times and then centrifuged for 2–5 min at 4 °C at 15,871 × g to remove any insoluble material. Fifty microliters of α-KG standard or cell sample was added to the well of 96-well plates. For each α-KG standard or cell sample, the reaction mix (50 μl) was added and incubated for 30 min at 37 °C and protected from light. The 96-well plates were measured on Spectra MRTM microplate spectrophotometer (Dynex Technologies) at OD570 nm.