Figure 6.

The transduction efficiency of scramble shRNA or NOX4 shRNA expression in hippocampus and reduced NOX4 expressions by NOX4 knockdown in 6-OHDA injected mice. (a) Establishment of U6 promoter-based NOX4 shRNA/AAV vector. The U6 promoter-driven shRNA expression system was established in the AAV vector. EGFP expression is separately controlled by a CMV promoter as a marker for the transduction efficiency. The NOX4 shRNA sequence was designed based on the siRNA sequence (boxed red nucleotides). (b) Representative photographs of tissue sections expressed EGFP (green) from mice hippocampal DG taken from mice injected with Scb shRNA/AAV2 or NOX4 shRNA/AAV2 and PBS (sham control) or 6-OHDA injection. To confirm NOX4 knockdown efficiency, NOX4 (red) expression was determined by NOX4 immunostaining in the hippocampal DG of 6-OHDA injected mice with shRNA/AAV2 or NOX4 shRNA/AAV2. (c) EGFP and NOX4 expression levels were quantified using Quantity One software. The results are expressed as a percentage of control. Data are shown as the mean ± SEM. n = 6. ### p < 0.001 vs. Scb shRNA 6-OHDA. (d and e) NOX4 knockdown efficiency in the hippocampal DG was also verified by both western blot analysis and RT-PCR performed after Scb shRNA or NOX4 shRNA injection. n = 6 per group. Expression levels were quantified using Quantity One software. The results are expressed as a percentage of control. Data are shown as the mean ± SEM. *** p < 0.001 vs. Scb shRNA control; ### p < 0.001 vs. Scb shRNA 6-OHDA. (f) NOX enzyme activity was measured in lysates of hippocampal tissue at 4 weeks after 6-OHDA treatment in Scb shRNA/AAV2 or NOX4 shRNA/AAV2-injected mice. n = 6 per group. * p < 0.01 vs. Scb shRNA control; # p < 0.05 vs. Scb shRNA 6-OHDA. Scb shRNA, scrambled shRNA; scale bars = 100 μm.