Effect of cocaine on c-fos mRNA levels and AP-1 activity. (A) Representative ethidium bromide-stained gel showing amplification of c-fos (upper band) and β-actin (lower band) transcripts obtained from RNA extracted from control and cocaine-treated (100 μM RIT) PC12 cells, and harvested 0.5, 1, or 2 h after the last of three intermittent cocaine treatments. Qualitative assessment indicated that compared to control, cocaine exposure increased levels of c-fos mRNA at all of the time-points examined. (B) Quantitative PCR (qPCR) analysis of c-fos mRNA levels relative to β-2 microglobulin in control and cocaine treated cells. Relative to control, 100 μM of RIT with cocaine significantly increased c-fos mRNA levels [F(1,21)=11.41, p<0.01]. (C) PC12 cells were transiently transfected with a luciferase-reporter plasmid containing 7 AP-1 transcription factor binding sites prior to treatment with 50 ng/mL NGF or cocaine (10, 100, or 500 μM SCT or RIT) for 6 h. Luciferase activity in each sample was normalized to its corresponding β-gal activity. Relative to control, there was a significant increase in luciferase activity in PC12 cells treated with NGF, or 10 or 100 μM RIT with cocaine. Results are representative of at least 5 independent experiments and data are presented as the mean ± SEM (*p<0.05, **p<0.01).