Figure 3
Evident Autophagy Was Observed in Iso-GNA Treated 549 cells.
(A) LC3 expression in A549 cells. Different concentrations of iso-GNA were incubated with A549 cells for 24 h. and 3-MA (2 mM) was treated as an autophagy inhibitor. (B) SQSTM1(p62) expression in A549 cells. Different concentrations of iso-GNA were incubated with A549 cells for 24 h. (C) 10 μM iso-GNA were treated with A549 cells(expression RFP-LC3)for 24 h. RFP-LC3 dots was observed by Leica aser-scanning spectrum confocal system. (D) 10 μM iso-GNA were treated with A549 cells for 24 h, then cells were subjected to transmission electron microscopy to observe double-membraned vacuolation (arrows) (E) Various concentrations of iso-GNA were treated with A549 cells for 24 h. then Beclin 1, Atg7 and Atg12-Atg5 complex expression level were detected by western blot. (F) A549 cells treated with 2 μM rapamycin, 10 μM iso-GNA with or with out 4 mM 3-MA for 24 h. Then LC3 immunofluorescence image in A549 cells were detected by a fluorescence microscope. Data of three independent tests were shown as means ± s.d. *p < 0.5; **p < 0.01, in comparison with the untreated group. The whole images of western blots are given in Supplementary Figure S7.
