Figure 6

P2-promoter activity in DRG neurons is decreased by Sp1-siRNA and Sp1 and Sp4 siRNA also block P2 promoter activity in NGF treated PC12 cells. Co-transfection of Sp1-siRNA with the 0.4 kb P2-promoter construct resulted in a significant decrease in promoter activity when compared with co-transfection of the scrambled (scr) siRNA control whereas Sp4-siRNA co-transfection failed to show a decrease (A). In contrast, both Sp1-siRNA and Sp4-siRNA co-transfection experiments showed a significant decrease in NGF treated PC12 cells (B). Primary cultures of NGF-treated dorsal root ganglion (DRG) neurons were transfected with either (pGL-E) empty luciferase reporter plasmid; ( 0.4 kb + pBS) Luciferase reporter containing the P2-promoter plus empty siRNA vector pBS/U6; (0.4 kb + siRNA-Sp1) 0.4 kb plus siRNA construct containing the Sp1 directed hairpin encoding Sp1 nucleotides 881-901; (0.4 kb + siRNA-Sp4) 0.4 kb plus siRNA construct containing the Sp4 directed hairpin encoding Sp4 nucleotides 1551-1571. (siRNAs were a gift from G. Gill Lab, Tufts, Boston) [16] . The presence of the scrambled siRNA control plasmid reduced the expected promoter activity of the 0.4 kb reporter plasmid in DRG. Co-transfection of the Sp1 cDNA in PC12 cells (0.4 kb + Sp1) directed a further increase in P2-promoter activity that was significantly reversed by co-transfection of the Sp1-siRNA construct. Error bars SEM (n = 3) triplicate measures. Significant differences: ANOVA (*) p < 0.05; (***) p < 0.001.