Fig 1: SOX12 expression positively correlates with GLS, GOT2, ASNS, and HIF-1a expression in CRC.a Representative images of IHC staining for SOX12, HIF-1a, GLS, GOT, and ASNS in human CRC tissues. b Analysis of correlations between SOX12 expression and GOT2 and ASNS expression in human CRC tissues. c Kaplan–Meier analysis of the associations between HIF-1a expression and the recurrence and overall survival of patients with CRC (upper panel). Kaplan–Meier analysis of the associations between concurrent SOX12 and HIF-1a expression and the recurrence and OS of patients with CRC (lower panel). d Kaplan–Meier analysis of the associations between GLS expression and the recurrence and OS of patients with CRC (upper panel). Kaplan–Meier analysis of the associations between simultaneous SOX12 and GLS expression and the relapse and OS of patients with CRC (lower panel). e Kaplan–Meier analysis of the associations between GOT2 expression and the relapse and OS of patients with CRC (upper panel). Kaplan–Meier analysis of the associations between simultaneous SOX12 and GOT2 expression and the relapse and OS of patients with CRC (lower panel). f Kaplan–Meier analysis of the associations between ASNS expression and the relapse and OS of patients with CRC (upper panel). Kaplan–Meier analysis of the associations between simultaneous SOX12 and ASNS expression and the relapse and OS of patients with CRC (lower panel)
Fig 2: 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
Fig 3: GLS, GOT2, and ASNS are essential for SOX12-mediated CRC cell proliferation and metastasis.a Western blotting analysis of GLS, GOT2, and ASNS levels in the indicated cells. b In vivo tumorigenesis assays showed that GLS, GOT2, and ASNS knockdown abolished SOX12-mediated tumorigenesis. The growth curves, tumor weights, and percentages of Ki67-positive cells in the indicated groups are shown. c In vivo tumorigenesis assays showed that GLS, GOT2, and ASNS overexpression attenuated the increase in CRC cell tumorigenesis induced by SOX12 knockdown. The growth curves, tumor weights, and percentages of Ki67-positive cells in the indicated groups are shown. d The BLI intensity of lung metastases in the different groups was traced for 9 weeks after implantation. The bioluminescence intensity at the indicated time points is presented as the total photon flux. e Incidence of lung metastasis in the indicated groups of nude mice. f Overall survival times in the indicated groups of nude mice. g Numbers of lung metastatic nodules. h Representative images of H&E-stained lung tissues from the different groups. The scale bars represent 200 µm (upper panel) and 50 µm (lower panel). *P < 0.05 compared with the control. The data are presented as the mean ± SD
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