Fig. 5: The production of NADPH, purine, hexosamine biosynthesis pathway (HBP) metabolites in SOX4 knockout cells or cells transfected with Scramble or si-STAT6.

A Cytoplasmic and Mitochondrial One-Carbon Folate Cycle: Folate is first reduced to tetrahydrofolate (THF), which can be converted to 5,10-methylene-THF (CH2-THF) through enzymatic reactions. In the mitochondria, the bifunctional enzyme MTHFD2 (highlighted in pink) catalyzes the conversion of CH2-THF to 10-formyltetrahydrofolate (¹⁰CHO-THF), a reaction that requires NAD + /NADP+ and exhibits dehydrogenase activity. This conversion is crucial for maintaining redox balance, especially via the GSH/GSSG system. The ¹⁰CHO-THF produced is then converted to formate by MTHFD1L and transported to the cytoplasm. In the cytoplasm, MTHFD1 regenerates ¹⁰CHO-THF from formate, which is essential for purine biosynthesis. Additionally, in the cytoplasm, CH2-THF serves as a substrate for thymidylate synthase (TYMS), which catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), critical for pyrimidine synthesis [40, 60]. This schematic was created with BioRender.com. B, C The metabolites abundances of the NADH, NAD, purine, uridine metabolites were analyzed by LC-MS in Hep3B and SOX4^−/− cells (B), Hep3B Scramble and Hep3B STAT si transfected cells (C). The metabolites abundances were normalized to control (Hep3B or Hep3B Scramble as one-fold). The corresponding metabolites abundances in SOX4^−/− or STAT6 si transfected cells was normalized to control and presented as a relative fold change. Data represent the mean ± SD from three independent experiments. (*P < 0.05, **P < 0.01, ***P < 0.001). D The levels of NADP+ and NADPH were measured in Hep3B and SOX4^−/− cells using a commercial kit (Abcam). The NADP/NADPH ratio was calculated as (NADP + - NADPH)/NADPH. E Hep3B cells transfected with scramble or STAT6 si were analyzed for NADP+ and NADPH levels using the Abcam kit. Data represent the mean ± SD from three independent experiments. (ns: non-significant, *P < 0.05, **P < 0.01).