Fig. 4

LPS enhances glycolysis and cell movements relying on HK3. a Effect of LPS on glucose uptake in RKO and DLD1 cells. Cells were cultured in glucose-free medium and treated with or without 1μg/ml LPS. After 24 h, cells were incubated with a fluorescent glucose derivative 2-NBDG for 30 min. Integrate optical density of fluorescence was detected using fluorescence microscopy and calculated by Image Pro Plus 6.0. Representative images from three independent experiments were shown. b Cells were cultured in glucose-free DMEM containing 10% FBS and 5mM glucose with or without LPS treatment. Cell medium supernatants were collected at 0, 1, 2, 4, 8, 16, and 24 h after LPS incubation. The concentration of glucose and lactate in cell medium supernatants was determined by glucose assay kit and lactic acid assay kit, respectively. c Cells were stimulated with 1μg/ml LPS for 24 h. Protein expression were determined by western blotting. Among several key glycolytic enzymes, only HK3 expression was obviously upregulated upon LPS treatment. d Scatter plot and spearman correlation analysis of HK3 and IL-1β mRNA expression levels in TCGA database (left) and our data (right) of CRC respectively. Logarithmic transformed data were used in our tissue samples. ρ was Spearman’s rank correlation coefficient. e Cells were transfected with non-targeting siRNA (siNC) or siRNA-Caspase-1 for 48 h, then treated with 1μg/ml LPS for 24 h. The effects of caspase-1 on HK3 expression with or without LPS treatment were examined by western blotting. f Cells were pretreated with 3μM caspase-1 inhibitor (Ac-YVAD-CHO) for 3 h, and then stimulated with LPS. Protein expression was determined by western blotting. g Cells that transient transfected with siRNA-HK3 or non-targeting siRNA (siNC) were treated with or without LPS. The initial glucose concentration was 5mM in culture medium. Glucose consumption and lactate production were detected by glucose assay kit and lactic acid assay kit in the indicated times respectively. h Transient siRNA transfection was performed by transfecting cells with 50nM control siRNA or siRNA-HK3 for 48 h, then treated with 1μg/ml LPS for 24 h. Transwell assay was used to detect cell migration and invasion ability; quantification of OD value was measured with microplate reader at 570 nm. Data are presented as mean±SEM. *P<0.05, **P<0.01, ***P<0.001