FIGURE 1.

Knockdown, proliferation, and viability of Ras-3T3 cells depleted of several glycolytic enzymes. A, relative knockdown of glycolytic enzymes in Ras-3T3 cells. Cell extracts harvested 4 days after transfection were assayed for the indicated enzyme activity with and without substrate, and the no substrate control was subtracted before calculation of units/mg protein in each lysate. Relative specific activity was normalized to the specific activity of mock-transfected cells for each enzyme. Mock transfection (black) and siRNA to the indicated enzyme (white) are plotted. Experiments were done ≥3 times, and error bars are expressed as S.E. Mock transfection values used for normalization were 0.027 ± 0.003 (aldolase), 0.23 ± 0.04 (GAPDH), 1.3 ± 0.17 (triose-phosphate isomerase; TIM), and 0.12 ± 0.02 (enolase) units/mg. B, cells were plated at 2.5 × 10⁴/35 mm dish, and at the indicated time points after transfection, the number of cells for each treatment was determined using a hemocytometer and plotted for mock-transfected cells (●), and cells were transfected with aldolase siRNA (○), GAPDH siRNA (■), triose-phosphate isomerase siRNA (□), or enolase siRNA (×). Error bars represent 1 S.D. C, mock (♦) and aldolase siRNA-transfected (♢) cells were stained with crystal violet each day after transfection and plotted versus time. Values are normalized to 1 day post-transfection values for each treatment. Error bars are represented as S.D. D, mock (♦) and aldolase siRNA-transfected (♢ cells were assayed for proliferation using MTS each day after transfection. The absorbance reading at 490 nm for each treatment each day is plotted versus time. Error bars are represented as S.D. E, cells treated as in B were subjected to trypan-blue exclusion assay. Live cells were counted, normalized to total number of cells and plotted as fraction of viable cells for each treatment. Symbols and error bars are as described in B.