Fig 2: RBX1 is overexpressed in ATC and closely correlated with poor prognosis in patients. A, B RBX1 mRNA expression levels in the ATC tissues together with the normal tissues adjacent to the tumor were investigated by qRT-PCR. **P < 0.01. C, D Expression levels of RBX1 protein in ATC tissues and the normal tissues adjacent to the tumor were investigated by western blotting. **P < 0.01. E RBX1 protein expression in the ATC tissues and the normal tissues adjacent to the tumor were determined using immunohistochemistry. Scale bar, 50 μm. F, G RBX1 protein and mRNA levels in ATC and PTC cell lines. ***P < 0.001. H Kaplan–Meier curves were used to visualize the overall survival of both low and high RBX1 expression of ATC patients. ***P < 0.001

Fig 3: RBX1 promotes ATC progression by upregulating PKM2 expression. A, B qRT-PCR and western blotting were performed for measuring the PKM2 and RBX1 expression. C Western blotting was performed to determine the PKM2 and RBX1 expression in various groups. D Quantification of transwell assay in various groups. *P < 0.05, **P < 0.01. E Quantification and representative images of the lung metastases in various groups of nude mice (n = 6). F Cellular glucose consumption, G6P levels, ATP levels, and lactate generation in the specific groups. *P < 0.05, **P < 0.01. G, H Measurement of OCR and ECAR in the specific groups. *P < 0.05. I PKM2 and RBX1 expression levels in various groups were determined using western blotting. J. Quantification of transwell assay in diverse groups. *P < 0.05, **P < 0.01. K Quantification and representative images of the lung metastases in various groups of nude mice (n = 6). L Cellular glucose consumption, G6P levels, ATP levels, and lactate generation in the specific groups. *P < 0.05, **P < 0.01. M, N ECAR and OCR were measured in the indicated groups. *P < 0.05, **P < 0.01

Fig 4: RBX1 facilitated the proliferation and migration of ATC cells. A Western blotting was performed to identify the RBX1 expression levels in CAL62 and KMH-5M cells stably transfected with shRBX1 plasmid. B RBX1 expression levels in the 8305C cells stably transfected with HA-RBX1 plasmid were measured using a western blot. C Transwell assays of CAL62 cells transfected with shRBX1 plasmid. **P < 0.01. D Transwell assays of 8305C cells transfected with HA-RBX1 plasmid. **P < 0.01. E EdU assays of CAL62 cells transfected with shRBX1 plasmid. **P < 0.01. F EdU assays of 8305C cells transfected with HA-RBX1 plasmid. **P < 0.01. G, H H&E staining of the sections of metastatic nodules in the lungs embedded with paraffin. *P < 0.05, **P < 0.01

Fig 5: RBX1 promotes ATC progression by enhancing the Warburg effect. A Cellular glucose consumption, G6P levels, ATP levels, and lactate generation in CAL62/shRBX1 cells. *P < 0.05. B Cellular glucose consumption, G6P levels, ATP levels, and lactate generation in 8305C/HA-RBX1 cells. *P < 0.05. C, D ECAR data showing the glycolytic rate and capacity in CAL62/shRBX1 cells. E, F ECAR data showing the glycolytic rate and capacity in 8305C/HA-RBX1 cells. G, H OCR results showing basal respiration and maximum respiration in CAL62/shRBX1 cells. I, J OCR results showing basal respiration and maximum respiration in 8305C/HA-RBX1 cells. K, L Lactate generation by 8305C/p-RBCK1 or CAL62/shRBX1 cells in the presence of 2-DG. *P < 0.05, **P < 0.01. M, N Role of 2-DG in the invasion and migration of 8305C/p-RBCK1 or CAL62/shRBX1 cells. *P < 0.05, **P < 0.01. O–Q Culturing 8305C cells in a medium with galactose but without glucose abolished the impact of RBX1 overexpression on cell invasion and migration, NS = No Significant