Fig 1: Evaluation of the levels of GLUT1 and O-GlcNAc in HCC and their clinical significance. (A) Immunohistochemistry technology was used to assess the protein levels of ACSL4, O-GlcNAc and GLUT1 in HCC tissues and adjacent normal tissues (Scale bar = 100 µm). (B–D) Pearson correlation analysis of the correlations between the levels of ACSL4, O-GlcNAc and GLUT1 in HCC tissues. (E, F) Kaplan-Meier analysis of the relationship between GLUT1/O-GlcNAc levels and the overall survival of patients with HCC.
Fig 2: ACSL4 was overexpressed in HCC tissue samples and cells. (A) The Oncomine database was used to retrieve the different expression patterns of ACSL4 in HCC or normal liver. (B, C) The mRNA and protein content of ACSL4 in the human normal liver cell line QSG-7701 and the HCC cell lines Huh-7, HLE, SK-HEP-1, BEL-7402 and HCCLM3 were determined by RT-PCR and western blotting, respectively. (D, E) The mRNA and protein content of ACSL4 in HCC tissues and normal tissues were detected by RT-PCR and western blotting assays. (F) Immunohistochemistry was used to detect ACSL4 protein expression in HCC tissues and normal tissues (Scale bar = 100 µm). (G) Kaplan-Meier analysis of the relationship between ACSL4 expression and the overall survival of patients with HCC. (*P<0.05, **P<0.01).
Fig 3: ACSL4 promoted HCC cell proliferation and repressed cell apoptosis via activating mTOR signalling. Huh-7 and SK-HEP-1 cells were transfected with si-NC, si-ACSL4, OE-NC or OE-ACSL4, with or without rapamycin treatment, and then the following assays were carried out. (A, B) RT-PCR and western blotting assays were carried out to assess the expression levels of ACSL4 at the mRNA and protein levels, respectively (*P<0.05, **P<0.01, si-ACSL4 group compared with si-NC group; #P<0.05, ##P<0.01, OE-ACSL4 group compared with OE-NC group). (C, D) The expression levels of mTOR and p-mTOR were detected by using a western blotting assay. (E, F) CCK-8 assay was used to detect cell proliferation (*P<0.05, compared with the control group; #P<0.05, compared with the OE-ACSL4 group). (G) Flow cytometry assay was used to test cell apoptosis (*P<0.05, compared with the control group; #P<0.05, compared with the OE-ACSL4 group).
Fig 4: Evaluation of the effects of the ACSL4/GLUT1 axis on cell proliferation, apoptosis and tumorigenesis in Huh-7 and SK-HEP-1 cells. (A, B) The mRNA and protein expression levels of GLUT1 were determined by RT-PCR and western blotting assays after cells were transfected with sh-GLUT1 or sh-NC, respectively (*P<0.05, **P<0.01, compared with the sh-NC group). Next, Huh-7 and SK-HEP-1 cells were transfected with OE-ACSL4 and/or sh-GLUT1 and subjected to the following assays. (C–E) Western blotting assays were used to assess the levels of O-GlcNAc, ACSL4 and GLUT1. (F, G) CCK-8 assay was carried out to test cell proliferation. (H) Flow cytometry assay was used to determine cell apoptosis. (I) An in vivo xenotransplantation assay was used to assess the effects of the ACSL4/GLUT1 axis on the tumour formation ability of Huh-7 and SK-HEP-1 cells. (C–I: *P<0.05, compared with control group; #P<0.05, compared with OE-ACSL4 group).
Fig 5: ACSL4 upregulation enhanced the stability of GLUT1 protein and reduced its ubiquitination. (A) After 12 hours of cell transfection with si-ACSL4 or OE-ACSL4, Huh-7 cells were treated with CHX (100 µg/ml) for 0, 1, 2, 4, 8 or 24 hours, and the western blotting assay was performed to detect GLUT1 expression. (B) An IP assay was used to detect the interaction between Ub and GLUT1 proteins after Huh-7 cells were transfected with si-ACSL4 or OE-ACSL4. (C) After 12 hours of cell transfection with si-ACSL4 or OE-ACSL4, SK-HEP-1 cells were treated with CHX (100 µg/ml) for 0, 1, 2, 4, 8 or 24 hours, and the western blotting assay was performed to detect GLUT1 expression. (D) IP assay was used to detect the interaction between Ub and GLUT1 protein in SK-HEP-1 cells. (*P<0.05, si-ACSL4/OE-ACSL4 group compared with control group).
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