Figure 4.

Cell-based and in vivo effects of small molecule ΔFosB regulators. (a) ΔFosB regulators alter reporter activity in cell-based transactivation assays. Neuro2A cells were cotransfected with a luciferase reporter gene (4xAP-1/RSV-Luc) and one of the following: ΔFosB, ΔFosB, and JunD, or the pcDNA 3.1 vector (to measure endogenous AP-1 transcriptional activity). Transactivation of the reporter gene was monitored after 48 h of incubation with 50 μM C1, C2, or C7, 25 μM C4, 12.5 μM C6, or 0.1% DMSO as a control. The change in luciferase signal is expressed as luciferase units per total cellular protein (LU/μgr). (b) ΔFosB regulators alter transcription of a ΔFosB target gene in mice. Real-time quantitative PCR was performed on triplicate samples of RNA isolated from the nucleus accumbens to evaluate levels of mRNA for ΔFosB target genes, GluR2 and cdk5, following 14 days administration of C2 (7 mice), a structurally related but inactive analogue (Chembridge 5996481, 8 mice) or vehicle (6 mice) in the nucleus accumbens. During the later 7 days of treatment, the animals additionally received an injection of 10 mgr/kg weight cocaine. Data (means ± SEM for each group of mice) were analyzed using an unpaired two-tailed t test with 95% confidence interval to reveal statistically significant differences in mRNA levels compared to the vehicle (p < 0.05).