Fig. 5.

Sp4 and its target genes are all regulated by neuronal activity in N2a cells. A. KCl-induced depolarization significantly increased mRNA levels of Sp4, 3 mitochondrial-encoded COX subunits, 3 mitochondrial transcription factors, SURF1, and 10 nucleus-encoded COX subunits. In the presence of Sp4 shRNA, however, KCl was not able to up-regulate the levels of all transcripts. B. TTX-induced impulse blockade led to reduced levels of Sp4, 3 mitochondrial-encoded COX subunits, 3 mitochondrial transcription factors, SURF1, and 10 nucleus-encoded COX subunits. Over-expression of Sp4, however, rescued all 13 COX subunits and 3 mitochondrial transcription factors as well as SURF1 from TTX-induced suppression N = 6 for each data point; *= P < 0.05; **= P < 0.01; ***= P < 0.001 when compared to either scrambled (A) or empty vector (B) controls. ### = P < 0.001 when compared to control + KCl (A) or control + TTX (B). C. Optical densitometric analysis of reaction product of cytochrome c oxidase histochemistry in N2a cells. KCl induced a significant increase in the relative activity of COX as compared to scrambled vector controls. However, no up-regulation was observed in cells transfected with Sp4 shRNA. N for optical densitometric (OD) analysis = 122 cells for control, 125 for control + KCl, and 128 for Sp4 shRNA + KCl. *** = P < 0.001 when compared to controls. ### = P < 0.001 when compared to control + KCl. No significance was found between Sp4 shRNA + KCl and controls. D. Three days of TTX treatment led to significant reduction of COX relative activity. However, in N2a cells transfected with Sp4 expression vectors, the activity was increased rather than down-regulated. N for OD analysis = 115 cells for empty vector controls, 162 for empty vector + TTX, and 207 for Sp4 over-expression + TTX. **= P < 0.01; ***= P < 0.001 when compared to controls. ### = P < 0.001 when compared to control + KCl.