Folic acid growth requirement of WT and mutant R113C PCFT transfectants. Exponentially growing PCFT transfectants were washed 3 times with PBS, transferred to folic acid-free RPMI-1640 medium supplemented with 10% dialyzed FCS (Invitrogen). After 10 days of deprivation in folic acid–free medium, cellular growth was arrested. Cells were then seeded in 24-well microplates (2 × 104 cells/well) containing increasing concentrations of folic acid ranging from 0.25 nM to 1 μM for WT PCFT transfectants and 10 nM to 3 μM for R113C PCFT as well as mock transfectants. After 7 days of incubation at 37°C, viable cell numbers were determined by hemocytometer count using trypan blue exclusion. A representative curve is shown from a total of 3 independent experiments. Actual cell numbers obtained for each folic acid concentration in the various transfectants are shown in the inset. The 100% control group used was the cell numbers obtained at the highest concentration of folic acid, ie, 3 μM for mock and R113C PCFT transfectants as well as 1 μM for folic acid for WT PCFT transfectants. Cell numbers obtained in these 100% control groups of mock, R113C PCFT, and WT PCFT transfectants were 3.7 × 105, 4.6 × 105, and 5.8 × 105, respectively.
