Cocaine-mediated induction of MCP-1 involves generation of ROS and Src kinase activation. (A) Cocaine-induced ROS generation in a time-dependent manner measured by laser-scanning microscopy. (B) BV-2 cells were treated with 10μM cocaine for the indicated time points (0-60 minutes) before incubation with carboxy-H2-DCF-DA and assessed for oxidative stress. Values are displayed as a ratio of the DCF fluorescent value to the Hoechst (nuclear stain) fluorescent value. A respiratory burst culminates after 15 minutes of stimulation. (C) BV-2 cells pretreated with apocynin (250μM) followed by stimulation with the cocaine for 30 minutes. Apocynin pretreatment resulted in abrogation of cocaine-induced respiratory burst. (D) Inhibition of NADPH oxidase by apocynin resulted in abrogation of cocaine-mediated induction of MCP-1. (E) Pretreatment of BV-2 cells with BD1047 and MβCD abrogated cocaine-induced ROS production. (F) Cocaine-induced Src phosphorylation in BV-2 cells. (G) Pretreatment with PP2 abrogated cocaine-induced ROS production. (H) Inhibition of the Src activity by Src inhibitor PP2 resulted in amelioration of cocaine-mediated induction of MCP-1. All the data are presented as means ± SD of 4 individual experiments. *P < .05; **P < .01 versus control group; #P < .05 versus cocaine-treated group.