FIGURE 6.

Activation of the CART promoter by forskolin was repressed by NRSF. A, schematic diagram of the reporter construct P-Luc-I that includes the CRE, pNRSE, and iNRSE elements. B, forskolin stimulation promotes CART luciferase activity. The pcDNA3.1 empty vector together with P-Luc-I (shown in Fig. 4A) and pRL-CMV were co-transfected into SK-N-SH cells. Then cells were stimulated with or without forskolin at the indicated concentrations. Twenty-four hours later, the cell lysates were prepared for luciferase activity analysis. Results are expressed as mean ± S.D. (n = 3). *, p < 0.05 (compare with the un-stimulated group). C, the SK-N-SH cells transfected as described in B were stimulated with forskolin of low or high concentrations (5 μm, 40 μm) for various times as indicated. The luciferase activity was measured. Results are expressed as mean ± S.D. (n = 3). *, p < 0.05. Based on these analyses, we selected 5 μm and 24 h stimulation of forskolin for the subsequent experiments described in Figs. 8 and 9. D, the pcDNA3.1 or pcDNA3.1-NRSF together with P-Luc-I and pRL-CMV were co-transfected into SK-N-SH cells. Then cells were stimulated with or without forskolin (5 μm, 24 h), and cell lysates were prepared for luciferase activity analysis. Results are expressed as mean ± S.D. (n = 3). E, quantitative analysis for CART mRNA was performed by real-time PCR. Each experiment was performed at least three times in triplicate. Data are presented as mean ± S.D. F, Western blot analysis was performed to measure the protein levels of CART.