Figure 5
METH treatment increases glutamate levels in proline-auxotrophic (CHO-K1) cells. CHO-K1 cells are proline auxotrophs that lack P5CS and PYCR2. (A) Western blot showing the absence of P5CS and PYCR2 in CHO-K1 cells (n = 3). β-actin was used as loading control. HEK293T cells that express both P5CS and PYCR2 was used as positive control. (B) Overexpression of P5CS in CHO-K1 cells. An expression construct of P5CS was generated using pcDNA 3.1. CHO-K1 cells were transfected with either pcDNA-empty vector or pcDNA-P5CS and post-transfection western blot was performed to confirm the expression of P5CS (n = 3). β-actin was used as a loading control. (C) Effect of METH treatment on glutamate levels in CHO-K1 in the presence and absence of P5CS expression. CHO-K1 cells were transfected with either pcDNA-empty vector or pcDNA-P5CS. Post-transfection the cells were treated with varying concentrations of METH under acute conditions. Glutamate levels were measured in the cell-free supernatants after 24 h (n = 3). Data represent the increase in glutamate levels over control untreated cells and are the mean ± SEM of at least three independent experiments. In (C), *p < 0.05, **p < 0.005 represents statistical comparison of untreated vs METH-treated cells shown in black bars, whereas *p < 0.05, **p < 0.005 represents comparison of METH-treated cells in the absence and presence of P5CS shown in gray bars.
