Research Paper Volume 12, Issue 7 pp 6415—6435

Figure 5. ES rescues Sirt3 expression to induce autophagy in VbP-treated THP-1 macrophages. (A) Western blot analysis of Sirt3 expression in macrophages treated with VbP and ES. (B) Analysis of the effects of ES and the Sirt3 inhibitor 3-TYP on Sirt3 expression. (C) DCFH-DA staining showing the effect of 3-TYP on ROS generation by macrophages (scale bar: 100 μm). (D) TEM images showing autophagosome assembly (red arrows) in VbP-treated macrophages exposed to ES (scale bar: 10 μm). (E) Visualization of AVOs by AO staining. The autophagy inducer Rapa (1 μM) was used as positive control. (F) DMC staining showing induction of autophagic vacuoles by ES in VbP-treated macrophages. This effect was blocked by pre-treatment with the autophagy inhibitor 3-MA (10 mM) (scale bar: 50 μm). (G) Immunofluorescence analysis of LC3 and Lamp2 expression indicating induction of autolysosome formation by ES and inhibition of this process by 3-TYP in VbP-treated macrophages (scale bar: 20 μm). (H) Western blotting analysis of the effects of ES and 3-TYP on ATG5, Sirt3, and LC3 expression in VbP-treated macrophages. n = 3; *P<0.05, **P<0.01, and ***P<0.001 vs. control cells; ^#P<0.05, ^##P<0.01, and ^###P<0.001 vs. VbP-treated cells.