Fig 2: HK2 promotes CSC stemness and tumor growth. (A-B) Immunoblotting analyses of H1048 (A) and H69 (B) cells with or without HK2 depletion were performed with the indicated antibodies. (C) The sphere formation ability of H1048 cells or H1048 cells with or without HK2 depletion in their first and the second passage was examined. Representative images were displayed (top), and the diameters and number of spheres were determined (bottom). Data shown are the mean ± SD (n = 3). ***, P < 0.001. Scale bars: 100 µm. (D-E) Immunoblotting analyses of H1048 (D) and H69 (E) cells with or without HK2 overexpression were performed with the indicated antibodies. (F) The sphere formation ability of H1048 cells with or without HK2 overexpression in their first and the second passage was examined. Representative images were displayed (top), and the diameters and number of spheres were determined (bottom). Data shown are the mean ± SD (n = 3). **, P < 0.01, ***, P < 0.001. Scale bars: 100 µm. (G) Immunodeficient mice (n = 10) were subcutaneously inoculated with an equal number (2 × 106) of H1048 cells with or without HK2 shRNA expression. Tumor sizes (top) and volumes (bottom) were measured and calculated. Data represent the means ± SD of ten mice per group. ***, P < 0.001. (H) IHC staining of mouse tumor tissues derived from H1048 cells with or without HK2 shRNA expression was performed with the indicated antibodies. Representative images were displayed (left), and the IHC scores were calculated (right). Data shown are the mean ± SD (n = 5). ***, P < 0.001. Scale bars: 100 µm. (I) Different numbers of H1048 cells with or without HK2 overexpression were subcutaneously injected into NOD/SCID mice (n = 10). Images of the tumors are shown on the left. Scale bar (right): 1 cm. Stem cell frequencies were estimated as the ratio 1/x with the upper and lower 95% confidence intervals (middle). ELDA analysis was performed (right). Abbreviations: HK2, hexokinase 2; CSC, cancer stem cell; SD, standard deviation; IHC, immunohistochemistry; ELDA, extreme limiting dilution analysis

Fig 3: HK2 regulated the effect of oxaliplatin in CRC cells. (A) GSEA was performed to detect enrichment of drug resistance-like pathways between HK2-high and HK2-low expression cohorts from TCGA. (B) GSEA was performed to detect enrichment of oxaliplatin resistance-like pathways between primary colorectal tumours and liver and lung metastases. (C) Cell viability of con-shRNA and HK2-shRNA RKO cells was assessed after oxaliplatin treatment at the indicated concentrations. (D) Flow cytometry assays were performed to analyse the apoptotic rate in con-shRNA and HK2-shRNA RKO cells after oxaliplatin (50 µM) treatment. (E) The apoptotic rate was analysed after 3-bp (50 µM) and/or oxaliplatin (50 µM) treatment and compared with that of untreated cells for 48 h by flow cytometry of the indicated cells. (F) Cell cycle analysis was performed after 3-bp (25 µM) and/or oxaliplatin (25 µM) treatment and compared with that of untreated cells for 48 h by flow cytometry in the indicated cells. The histogram represents the quantitative analysis. *p < 0.05; **0.001 < p < 0.01; ***p < 0.001. Data are represented as the mean ± SEM

Fig 4: Downregulation of miR-128-3p restored the si-CCNG1-induced effect on OC cells. (A and B) Cell viability was examined by MTT assay after transfection of si-CCNG1, si-CCNG1+anti-miR-128-3p or the corresponding controls. (C and D) Cell apoptosis was detected by flow cytometry. (E and F) Cell migration and (G and H) invasion were measured by Transwell assay. The examination of (I and J) glucose consumption and (K and L) lactate production was performed via glucose detection and lactic acid detection kits. (M and N) The protein expression of HK2 was determined by western blotting assay. *P<0.05. miR, microRNA; si-, small interfering; CCNG1, cyclin G1; OC, ovarian cancer; HK2, hexokinase 2.

Fig 5: GAS1 overexpression impaired cell proliferation, angiogenesis, migration, invasion, EMT, glycolysis and ROS homeostasis in bladder cancer cells. a. The effect of GAS1 on cell viability was verified by a CCK-8 assay. b. A colony formation assay showed that GAS1 impaired the colony-forming ability of T24 and EJ cells. c. Conditioned medium was collected from control or GAS1-overexpressing cells and used in a tube formation assay to evaluate angiogenesis. d. A wound healing assay indicated the effect of GAS1 overexpression on migration. e. GAS1 overexpression suppressed the migration and invasion of T24 and EJ cells, as determined by Transwell migration and Matrigel invasion assays. f-g Western blot and qRT-PCR analyses showing the expression levels of EMT markers in GAS1-overexpressing bladder cancer cells at the protein and mRNA levels, respectively. h-i The effects of GAS1 on glycolysis were determined by glucose uptake and lactate production assays. j-k PFKFB3, HK2 and GLUT1 levels in GAS1-overexpressing cells were evaluated by western blotting and qRT-PCR. l. GAS1 remarkably upregulated intracellular ROS levels in T24 and EJ cells. m. TCGA database analysis showed a lower expression level of GAS1 expression in bladder cancer tissue than in normal tissue. n. Immunohistochemical detection of GAS1 in cohort 2 (n = 30) further confirmed the dysregulation of GAS1 in bladder cancer. Scale bar: 200 μm. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group