Establishment of EGFP-LC3-HEK293 Stable Cell Line and the Role in Aβ-induced Autophagy

Abstract: In this study， an EGFP-LC3-HEK293 stable cell line has been established. The cells were incubated with 10 μmol/L of Aβ and the truncated peptides with various percentages of hydrophobic amino acids (Aβ40， Aβ42，Aβ1-11， Aβ12-24， Aβ25-35 and Aβ35-42) for 24 h. The expression of LC3 was detected by fluorescence microscopy and Western blot， cell viability was measured via MTT assays and the ultrastructural morphology was studied by transmission electron microscopy (TEM). As a result， the best concentration of G418 was 700 μg/mL for EGFP-LC3 stable clone screen. The quantitative immunofluorescence microscopy data showed that Aβ25-35， Aβ40 and Aβ42 induced autophagy significantly in EGFP-LC3-HEK293 stable cells. MTT assays showed much stronger cytotoxicity in Aβ42-treated group than in Aβ40 group whereas both peptides induced autophagy. LC3BII/I shifting which indicated LC3B lipidation was detected via Western blot. Aβ-induced autophagosomes could be observed by TEM. Our data suggests that autophagy can be induced by Aβ peptides and the truncated forms of hydrophobic amino acids fregment， but the autophagy induction is not correlated with the percentage of hydrophobic amino acids. Furthermore， Aβ42 is much more cytotoxic than Aβ40 during autophagy induction. Based on this study， our research lay the foundation for further mechanisms of autophagy in Alzheimer’s disease.

CSTR: 32200.14.cjcb.2015.07.0009