Figure 3.

SIRT7 deficiency inhibits Aβ-induced ROS generation. (A) Intracellular ROS levels were quantified by flow cytometry after SH-SY5Y cells were loaded with 10 μM CM-H2DCFHDA, pretreated with 1 mM NAC for 1 h, and then treated with 5 μM Aβ₄₂ for a further 3 h in the presence of 1 mM NAC. Histogram of DCF-DA intensity of a representative experiment. (B) Vertical lines indicate the mean fluorescence values with the control cells set as 1. The geometric mean fluorescence intensity (MFI) ± SEM of three independent experiments was analyzed. (C) SH-SY5Y cells were pretreated with 1 mM NAC for 1 h, and then incubated in the presence of 5 μM Aβ₄₂ and 1 mM NAC for a further 24 h. The intensity of cleaved caspase 3 was determined by Western blot analysis. (D) The value of cleaved caspase 3 was normalized to that of β-actin. (E) Flow cytometry analysis was performed on cells treated in the same condition as that in Figure 3C. Representative flow cytometry plots using annexin V-FITC/PI staining for apoptosis. (F) The percentage of total annexin V-positive cells was calculated. (G) Experimental scheme for analyzing Aβ₄₂-induced ROS in SIRT7 KD SH-SY5Y cells. (H) Aβ₄₂-induced ROS generation in control and SIRT7 KD SH-SY5Y cells was evaluated after they had been treated with Aβ₄₂ for 3 h. Intracellular ROS levels after DCF-DA loading were visualized by fluorescence microscopy. Scale bar, 50 μm. (I) Intracellular ROS levels were quantified by flow cytometry on cells treated in the same condition as that in Figure 3G. Histogram of DCF-DA intensity in a representative experiment. (J) The geometric MFI ± SEM of three independent experiments was analyzed. (K) Mitochondrial ROS levels were assessed by flow cytometry after the cells had been treated with Aβ₄₂ for 3 h and stained with 5 μM MitoSOX Red for a further 15 min. Histogram of MitoSOX Red intensity in a representative experiment. (L) The geometric MFI ± SEM of three independent experiments was analyzed. All data are shown as the mean ± SEM. Statistical significance was determined by two-way ANOVA with Tukey’s post hoc test. **** p < 0.0001. ANOVA N.S., p > 0.05.