FIGURE 10.

Down-regulation of LDH does not significantly affect the rates of glucose consumption and lactate generation by intact 4T1 cells. A, LDHA siRNA transfection significantly reduced the amount and activity of LDH. 4T1 cells were transiently transfected with LDHA siRNA or negative control siRNA for 72 h; cells were collected and lysed; the cell lysate was subjected for Western blot and enzyme assay for LDH. B, LDHA siRNA transfection did not significantly decrease glucose consumption and lactate generation. Cells were incubated in fresh 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. The detailed procedure of siRNA knockdown, Western blot, LDH enzyme activity assay, and determination of glucose and lactate is described under “Experimental Procedures.” Data are means ± S.D., n = 4.