Figure 4
METH treatment of P5CS knockout (P5CS-KO) SH-SY5Y cells significantly increases glutamate levels. The P5CS-KO SH-SY5Y cells were generated from wild-type SH-SY5Y cells using CRISPR/Cas9 system. Following which the P5CS-KO cells were treated with METH for 24 h and cellular lysates and cell-free supernatants were collected. (A) Western blot showing a lack of P5CS expression in P5CS-KO vs. WT SH-SY5Y cells (n = 3). β-actin was used as a loading control. (B,C) Effect of METH on glutamate in P5CS-KO cells. (B) The cell extracts were used to measure intracellular glutamate while (C) the cell-free supernatants were used to measure extracellular glutamate (n = 3). A marked increase in levels of both intracellular and extracellular glutamate was obtained in P5CS-KO cells after METH treatment. (D,E) Effect of acute METH treatment on PYCR2 in P5CS-KO cells. Representative western blot of PYCR2 in (D) following acute METH treatment (n = 3). β-actin was used as a loading control. (E) Densitometric analyses of PYCR2 normalized to β-actin. (F–H) Effect of acute METH treatment on GLS and vGlut1 in P5CS-KO cells. (F) Representative western blot of GLS and vGLUT1 in METH-treated P5CS-KO SH-SY5Y cells (n = 3). β-actin was used as a loading control. (G,H) Densitometric analyses of GLS and vGLUT1 western blot normalized to β-actin. Data are presented as the mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.005 represents statistical comparison of untreated vs METH-treated cells.
