FIGURE 11.

Down-regulation of HK and PFK1 significantly decreases glucose consumption and lactate generation by 4T1 cells. A, HK2 siRNA transfection significantly reduced the amount and activity of HK. 4T1 cells were transiently transfected with HK2 siRNA or negative control siRNA for 72 h; cells were collected and lysed; and the cell lysates were subjected for Western blot and enzyme assay for HK. B, HK2 siRNA transfection significantly decreased glucose consumption and lactate generation. Cells were incubated in culture medium for a further 5 h; the concentrations of glucose and lactate in culture medium were determined, and then glucose consumption and lactate generation were calculated. C, PFK1 siRNA transfection significantly reduced the amount and activity of PFK1. 4T1 cells were transiently transfected with PFK1 siRNA or negative control siRNA for 48 h; RNA was extracted and subjected to real time PCR determination of PFK1; enzyme assay for PFK1 activity was carried 72 h after siRNA transfection. D, PFK1 siRNA transfection significantly decreased glucose consumption and lactate generation. Cells were incubated in fresh culture medium for further 5 h; the concentrations of glucose and lactate in culture medium were determined, and then glucose consumption and lactate generation were calculated. The detailed procedure of siRNA knockdown, Western blot, enzyme activity assay, and determination of glucose and lactate is described under “Experimental Procedures.” Data are mean ± S.D., n = 4.