Figure 1.
Amyloid-β peptide 25–35 (Aβ25–35) inhibits the growth of SH-SY5Y cells.
Cell viability was examined by MTT assay in SH-SY5Y cells. Cells were treated with various doses of Aβ25–35 for 24 h (A) and 10 µM Aβ25–35 for various times (B).
Figure 2.
Aβ25–35 induces autophagy in SH-SY5Y cells.
(A) Western blot analysis of LC3 protein expression in SH-SY5Y cells treated with different doses of Aβ25–35 and quantification, β-actin was a loading control. (B) Immunofluorescence microscopy of punctate pattern of LC3 localization in SH-SY5Y cells treated with Aβ25–35. (C) Electron micrographs of SH-SY5Y cells treated with Aβ25–35 for 24 h. (D) Fluorescence microscopy of the formation of acidic vesicles after MDC staining in SH-SY5Y cells treated with Aβ25–35 for 4 h. (E)Western blot analyses of the protein expression of LC3 and p62 in cells treated with Aβ25–35 with and without chloroquine.
Figure 3.
Aβ25–35-induced growth inhibition of SH-SY5Y cells is mediated by autophagy.
(A) Annexin V/PI staining of apoptosis of SH-SY5Y cells treated with different doses of Aβ25–35 for 24 h. (B) Hoechst staining of apoptosis of SH-SY5Y cells treated with different doses of Aβ25–35 for 24 h. (C) Immunoblotting of Beclin 1 and LC3 expression with SH-SY5Y cell lysates. Cells were treated with 10 µM Aβ25–35 for additional 24 h after transfection with random siRNA or beclin 1 siRNA for 48 h. (D) MTT assay of cell viability of SH-SY5Y cells treated with 10 µM Aβ25–35 for 24 h after transfection with random siRNA or beclin 1 siRNA for 48 h.
Figure 4.
MLA alleviates the toxic effect of Aβ25–35 in SH-SY5Y cells.
MTT assay of cell viability of SH-SY5Y cells treated with (A) various doses of MLA for 24 h or (B) 10 µM Aβ25–35 with various doses of MLA.
Figure 5.
MLA inhibits Aβ-induced autophagy process in SH-SY5Y cells.
(A) Western blot analysis of the protein level of LC3 in SH-SY5Y cells treated with 10 µM Aβ25–35 with or without MLA. (B) Immunofluorescence microscopy of punctate pattern of LC3 localization in SH-SY5Y cells treated with 10 µM Aβ25–35 with or without MLA. (C) Fluorescence microscopy of the formation of acidic vesicles with MDC staining in SH-SY5Y cells treated with 10 µM Aβ25–35 with or without MLA. (D) Flow cytometry of the formation of acidic vesicles after MDC staining in SH-SY5Y cells treated with 10 µM Aβ25–35 with or without MLA.
Figure 6.
MLA inhibition of Aβ-induced autophagy is mediated by mTOR signal pathway in SH-SY5Y cells.
(A) After exposure to different doses of Aβ25–35, the level of phosphorylated p70S6K, a mTOR complex 1 substrate, was evaluated by western blot analysis. Signals were quantified by densitometry. (B) Western blot analysis of level of phosphorylated p70S6K after exposure to 10 µM Aβ25–35 with or without MLA.