Fig. 2

Protective autophagy induced by icotinib mediates icotinib resistance in EGFR-mutated NSCLC cells. A TEM depicted ultrastructural changes in NSCLC cells in response to icotinib (2 μM/4 weeks). Arrows point to autophagic vehicles. Scale bar: 1 μm. B PC-9/GR cells stably expressed mRFP-GFP-LC3 were treated with DMSO or 2 μM icotinib for 4 weeks, and were detected by laser confocal fluorescence microscopy. In the merged image, yellow puncta indicate autophagosomes, while red puncta indicate autolysosomes. Scale bar: 10 μm. C Immunoblotting assays assessed the expression of Beclin-1, VPS34, ATG14, ATG3, ATG7, LC3B and p62 in PC-9/GR and H1975 cells treated with icotinib or DMSO for 4 weeks. D-F Autophagy inhibitors CQ (10 μM/24 h), BafA1 (10 nM/24 h) and 3-MA (1 mM/24 h) were added to icotinib (2 μM/4 weeks) -exposed cells for 24 h. Autophagy analysis including immunoblotting detection of changes in cleaved PARP and LC3B conversion (D), and observations of mRFP-GFP-LC3 puncta (E) were observed in indicated cells. Scale bar: 10 μm. Cell apoptosis was analyzed using flow cytometry analysis (F). G-I Icotinib-exposed PC-9/GR and H1975 cells were transfected with siATG3, siATG7 and siBeclin-1. Autophagy analysis including immunoblotting detection of changes in cleaved PARP and LC3B conversion (G), and observations of mRFP-GFP-LC3 puncta (H) were observed in indicated cells. Scale bar: 10 μm. Cell apoptosis was analyzed using flow cytometry analysis (I). [**P < 0.01, ***P < 0.001, ns, no significance as compared with the control group. ###P < 0.001, NS, no significance as compared with the icotinib alone group]