Figure 7
The autophagy and cytotoxicity induced by afatinib are related to intracellular ROS. (A–E) H1975 and H1650 cells were treated by 10 μM afatinib in the presence or absence of 5 mM NAC for 48 h. (A) ROS assay kit was used to analyze intracellular ROS by measuring the DCF fluorescence intensity (*P < 0.05, **P < 0.01). (B) Cell viability was analyzed with MTT assay (*P < 0.05). (C) The levels of LC3-I/II were measured by western blot analysis. These blots were cropped with Photoshop CS6. (D) Western blot analysis was used to detect the level of PARP and Cleaved-PARP. These blots were cropped with Photoshop CS6. (E) H1975 and H1650 cells were co-stained with Cyto-ID ®Green Dye and Mito SoxTM Red Dye at 37 °C for 15 min, and detected by confocal microscopy. (F) After treated with 10 μM afatinib for 0, 6, 12, 18, 24 and 48 h, H1975 and H1650 cells were stained with Cyto-ID ®Green Dye and Mito SoxTM Red Dye at 37 °C for 15 min, then analyzed by confocal microscopy.
