Fig. 4. SOX12 regulates asparagine synthesis by transactivating ASNS, GLS, and GOT2 expression in human CRC.

a ASNS, GLS, and GOT2 are three key enzymes in asparagine synthesis. b, c After CRC cells were infected with LV-SOX12 or LV-shSOX12, GLS, GOT2, and ASNS levels were detected using qRT-PCR (b) and western blotting (c, d). After cotransfection of the luciferase constructs containing the (− 2046/ + 36) GLS, (− 3786/ + 102) GOT2, or (− 1191/ + 111) ASNS promoters with pCMV-SOX12, the relative luciferase activity was determined. e–g) Serially truncated and mutated GLS (e), GOT2 (f), and ASNS (g) promoter plasmids were cotransfected with pCMV-SOX12, and promoter luciferase assays were performed. h–j A ChIP assay revealed direct interactions between SOX12 and the GLS (h), GOT2 (i), and ASNS (j) promoters in CRC cells. k The levels of the indicated intracellular metabolites in SW480, Caco-2, SW620, and LoVo cells were analyzed using LC-MS/MS. *P < 0.05 compared with the control. The data are presented as the mean ± SD