Fig 2: Functional studies of BMI1 in HCC progression. (a) Kaplan-Meier survival analysis for overall survival and disease-free survival of the TCGA-LIHC patients based on BMI1 mRNA level. (b) Western blot analysis of BMI1 expression in 18 individual paired HCC tissues. (c–e) Cell proliferation assay was performed in HCC cells after PTC-209 treatment. (f) Colony formation assay was conducted in HCC cells treated with PTC-209. (****p < 0.0001) (g) Volcano plot for DEGs between BMI1 wild-type and BMI1 knockout mice liver tissues using GSE97172 dataset. (h) KEGG pathway analysis for DEGs identified in (g). (i) The correlation of BMI1 expression with immune infiltration level was analyzed in HCC tissues. (j) Coexpression analysis between the expression level of PD-L1, Galectin 9, HVEM, IDO, and BMI1 using TCGA-LIHC data.

Fig 3: Effects of silencing the Bmi-1 gene on glucose uptake and lactate levels in human gastric adenocarcinoma AGS cells. (a) Relative glucose uptake in AGS cells transfected with Bmi-1-specific siRNA (Bmi-1 siRNA), nonspecific siRNA as a control (NC), and without any treatment (Blank) as well as in human immortalized gastric epithelial GES-1 cells. The relative glucose uptake was significantly decreased in AGS cells transfected with Bmi-1 siRNA vs NC. ** indicates a significant difference between the two groups, P < 0.05; (b) relative lactate levels in AGS cells transfected with Bmi-1 siRNA, NC, and without any treatment (Blank), and in GES-1 cells. The relative lactate level was significantly lower in AGS cells transfected with Bmi-1 siRNA vs NC. ** indicates a significant difference between the two groups, P < 0.05.

Fig 4: Bmi-1 and GLUT1 protein expression levels in (GAC) tissues and normal gastric specimens. Immunohistochemistry and western blot analysis were performed to examine the Bmi-1 and GLUT1 protein expression levels in GAC specimens (n = 4) and matched histologically normal gastric specimens (n = 4). Representative images of immunohistochemical staining of Bmi-1 in (a) GAC and (b) noncancerous control tissues (magnification, 400×). The yellow-brown color denotes positive Bmi-1 expression that is mainly located in the nucleus (arrows). Representative images of immunohistochemical staining of GLUT1 in (c) GAC and (d) noncancerous control tissues (magnification, 400×). GLUT1 is mainly localized in the cell membrane (arrows); (e) Western blot analysis of Bmi-1 and GLUT1 protein expressions in four paired tissues of GAC and noncancerous control tissues. The relative Bmi-1 and GLUT1 protein levels were normalized to GAPDH; (f) quantification of the Bmi-1 and GLUT1 protein expression levels in three independent experiments on the basis of the immunohistochemical and western blot images. The levels of Bmi-1 and GLUT1 were significantly greater in AGC tissues vs matched adjacent noncancerous tissues (3.903 ± 0.9363 vs 1.613 ± 0.760 and 2.480 ± 1.276 vs 1.945 ± 0.230, respectively) (All P < 0.05). T, tumor tissues; P, paracancerous, noncancerous tissues.

Fig 5: Effects of silencing Bmi-1 on GLUT1 protein expression in human gastric adenocarcinoma AGS cells. (a) qPCR analysis of GLUT1 mRNA expression levels in AGS cells transfected with Bmi-1-specific siRNA (Bmi-1 siRNA), nonspecific siRNA as a control (NC), with no treatment (Blank), as well as in human immortalized gastric epithelial GES-1 cells; (b) Western blot analysis of Bmi-1 and GLUT1 protein expression in AGS cells transfected with Bmi-1 siRNA, nonspecific siRNA as a control (NC), with no treatment (Blank), as well as in GES-1 cells; (c) quantification of the Bmi-1 protein levels shown in the western blots. The relative Bmi-1 protein expression was normalized to GAPDH. * indicates a statistical difference between the two groups, P < 0.05; (d) quantification of the GLUT1 protein levels shown in the western blots. The relative GLUT1 protein expression was normalized to GAPDH. * indicates a statistical difference between the two groups, P < 0.05.