Figure 5.

SIRT7 deficiency inhibits Aβ-induced NOX4 protein expression. (A) Western blot analysis of NOX4 was performed when control and SIRT7 KD SH-SY5Y cells had been treated with Aβ₄₂ for 3 h. (B) The value of NOX4 was normalized to that of β-actin. (C) NOX4 mRNA expression was determined with the quantitative real-time RT-PCR analysis of cells treated in the same condition as that in Figure 5A. The value of NOX4 mRNA was normalized to that of 18S rRNA. (D) Experimental scheme for the effect of double KD of SIRT7 and NOX4 on Aβ₄₂-induced ROS and apoptosis. (E) Control and NOX4 KD SH-SY5Y cells transfected with either control siRNA or SIRT7 siRNA were treated in the presence of 5 μM Aβ₄₂ for 3 h. Intracellular ROS levels were evaluated by flow cytometry. (F) For quantification of intracellular ROS levels, the geometric MFI ± SEM of three independent experiments was analyzed. (G) Flow cytometry analysis was performed on cells treated in the same condition as that in Figure 5D. Representative flow cytometry plots using annexin V-FITC/PI staining for apoptosis. (H) The percentage of total annexin V-positive cells was calculated. (I) Western blot analysis of cleaved caspase 3 was performed on cells treated in the same condition as that in Figure 5D. (J) The value of cleaved caspase 3 was normalized to that of β-actin. All data are shown as the mean ± SEM. Statistical significance was determined by either one-way ANOVA with Tukey’s post hoc test or two-way ANOVA with Tukey’s post hoc test. N.S., not significant; **** p < 0.0001. ANOVA N.S., p > 0.05.