Fig 2: SIX1 activates the HBP pathway and increases O-GlcNAcylation in HCC. BEL7402 and BEL7404 cells were transduced with SIX1 overexpression or knockdown lentivirus, respectively. A The glucose levels were measured in the cell culture media of cells with SIX1 overexpression or knockdown B Glucose uptake levels were determined in the cell line with different SIX1 expressions. C The mRNA levels of GLUT1, HK2 and GFAT1 were analyzed with qPCR in cells with SIX1 overexpression or knockdown. D GLUT1, HK2 and GFAT1 were analyzed by western blotting in cells with SIX1 overexpression or knockdown. E O-GlcNAcylation was analyzed by western blot in HCC cells with SIX1 knockdown or overexpression. (The data were analyzed by Student's t-test (BEL7402) and one-way ANOVA (BEL7404).*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: HBP's status in melanoma cell lines. (A) Protein levels of total GFAT, (B) GFAT1, and (C) GFAT2 were measured by western blotting in WM983A (black bars) and WM852 (gray bars) cell lines. Quantification of protein levels in each cell line was normalized to ß-tubulin. (D) Total and relative GFAT activity was measured in cell lysates by a colorimetric assay. Global GFAT activity was normalized by global GFAT expression in order to isolate activity from expression levels. (E) Quantification of UDP-GlcNAc by cell number and representative chromatogram showing UDP-GlcNAc peak in the two cell lines. All experiments were performed with at least 3 biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.