Fig. 5

EOAI treatment activated autophagy. A Immunofluorescence of LC3. A549 and H460 cells were treated with EOAI (2 μΜ) for 24 h. Cells were then incubated with LC3 primary antibody (1:200, overnight at 4℃) and Alexa Fluor 488 Goat Anti-Rabbit IgG (H + L) secondary antibody (green) (1:500, 2 h at room temperature), respectively. The nuclei were stained by DAPI (blue) (5 μg/mL, 20 min at room temperature). Images were captured using fluorescence microscopy (magnification: 200). Representative images were shown. B Detection of the expression of LC3. A549 and H460 cells were treated with EOAI, and cell extracts were prepared for Western blotting analysis. GAPDH was used as the loading control. C Blockage of autophagy enhances EOAI-induced cell growth suppression of NSCLC cells. A549 and H460 cells were treated with 2 μmol/L EOAI alone or in combination with 3MA (10 mmol/L). Cell viability was measured using the CCK-8 assay. D Detection of the efficacy of EOAI alone or in combination with 3MA on colony formation on NSCLC cells. NSCLC cells were treated with EOAI alone or in combination with 3MA as indicated for 10 days and then fixed and stained. E Blockage of the autophagic response increased the EOAI-induced apoptosis of NSCLC cells. A549 and H460 cells were treated with 2 μmol/L EOAI alone or in combination with 3MA (10 mmol/L). Apoptosis was determined by the Annexin V-FITC/PI double-staining analysis. F Total PARP and cleaved PARP, and p-AMPK were detected by immunoblotting. GAPDH was used as the loading control. All data were representative of at least three independent experiments