Fig. 2. DHA-37 induces the autophagic cell death in non-small-cell lung carcinoma A549 cells.

a, b A549 cells were respectively pre-treated with CQ, 3-MA, LY294002, or Z-VAD-FMK at indicated concentrations for 2 h, then 10 μM DHA-37 was added and co-incubated for 48 h. MTT assay was used to detect the cell viability. c A549 cells were transfected with control siRNA or siRNA targeting ATG7. The knockdown of ATG7 was confirmed by western blotting. ATG7 downregulated A549 cells were treated with 10 μM DHA-37 for 48 h, and the cell viability was measured by MTT assay. d A549 cells were treated with DHA-37 at indicated concentrations for 24 h before cell lysis. Cell extracts were analyzed by western blotting. e A549 cells were treated with 10 μM DHA-37 at indicated time before cell lysis. Cell extracts were analyzed by western blotting. f A549 cells were pre-treated with 10 μM CQ for 2 h, then 10 μM DHA-37 was added and coincubated for 24 h. Cell extracts were analyzed by western blotting. g A549 cells were cultured with indicated concentrations of DHA-37 for 24 h. Then, the expression levels of p62 were analyzed by western blotting. h pcDNA3.1-LC3-GFP and pcDNA3.1 were transfected into A549 cells and then cells were treated with 10 μM DHA-37 and DHA, respectively, for 24 h. The formation of vacuoles containing GFP-LC3 (dots) was examined by confocal microscopy. i Cells were treated with 10 μM DHA-37 or DHA, respectively, for 24 h, cells were then fixed and the images were acquired with scanning electron microscope. j pcDNA3.1-LC3-GFP transfected A549 cells were treated with DHA37 for 24 h in the presence or absence of bafilomycin A1. The formation of vacuoles containing GFP-LC3 (dots) was examined by confocal microscopy. The endogenous expression levels of LC-3 were analyzed by western blotting. k The data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01