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. 2020 Dec 19;11(5):1227–1245. doi: 10.1016/j.apsb.2020.12.013

Figure 10.

Figure 10

MA is a novel SIRT3 activator that induces MCF-7 cell autophagy. (A) Chemical structure of a new SIRT3 activator, 1-methylbenzylamino amiodarone (MA). (B) Molecular docking of MA and SIRT3. (C) The cellular SIRT3 deacetylase activity of MA was determined by SIRT3 activity assay kit using cellular extracts obtained from MCF-7 cells. (D) The expression levels of SIRT3 as well as acetylated MnSOD2 at K68 and K122 were determined by Western blot. The relative expression level of SIRT3, acetylated MnSOD2 at K68 and K122 was quantified by normalization to β-actin expression. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. control. (E) Representative immunofluorescence images of LC3 puncta in MCF-7 cells transiently expressing GFP-mRFP-LC3 plasmid followed by treatment with 20 μmol/L MA. Scale bar = 20 μm. (F) Representative immunofluorescence images of SQSTM1/P62 expression and location in MCF-7 cells treated with 20 μmol/L MA. Scale bar = 10 μm. (G) Western blot analysis of ATG4B, ATG5, SQSTM1/P62, beclin-1 and LC3 in MCF-7 cells treated with 20 μmol/L MA for 6, 12, or 24 h. Relative ATG5, beclin-1 and LC3 II expression were quantified by normalization to β-actin expression; ns, no significance; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. (G) Would healing assay of MCF-7 cells after MA treatment. Percent wound closure was quantified by Image J software; ∗∗∗P < 0.001. (H) Differential expression of E-cadherin in MA-treated MCF-7 cells. Cells were counterstained with DAPI. Relative E-cadherin intensity was quantified by Image J software; ∗∗∗P < 0.01. Scale bar = 10 μm.