Figure 4

METH induces oxidative stress in NPCs. NPCs maintained under proliferative conditions were exposed to various concentrations of METH. Numbers following "M" refer to concentration of METH in uM. 3-NP, DMNQ, and STS are inducers of oxidative stress and were used as positive controls. A. NPCs exposed to METH for 4 hrs were loaded with Carboxy-H2 DCFDA. ROS formation (green) is far greater in cells exposed to METH (250 and 500 uM) than control. B. Nitrite production from supernatants of NPCs was assessed via the Griess reaction. Values were normalized to supernatants from untreated NPCs.C. 3-nitrotyrosination of cell lysates increases in a dose-dependent manner in NPCs exposed to METH as demonstrated by quantification of slot blots, suggesting increased oxidative and/or nitrosative stress. D. Lysates from untreated NPCs (Control) or those exposed to STS, METH 250 uM (M250) and 1000 uM (M1000) for 24 hours were subjected to western blotting with 3-nitrotyrosine antibody. Several bands, including those of apparent molecular mass 10, 25, 30, 32, 45, and 90 kDa, were preferentially nitrotyrosinated in NPCs exposed to METH (250 and 1000 uM) as compared to control cells. * p <0.05, ** p <0.01 ANOVA.