The inhibition of CX-4945-induced autophagy led to decreased apoptosis.
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A, PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h and then were fixed with methanol, immunostained with anti-LC3 (red), anti-EGFR (green), and DAPI (blue), and analyzed by confocal microscopy to determine the intracellular localization of EGFR. B, The suppression of Atg7 by siRNA treatment was detected by Western blot analysis. PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h in the presence or absence of 3MA (2 mM) and Atg7 siRNA (100 nM). The modulation of EGFR was detected by Western blot analysis. C and D, Cells were treated with drugs as in Fig. 2 under the presence or absence of 3MA and Atg7 siRNA. Cleavage of PARP-1 and caspase-3 was shown by Western blot analysis. Apoptosis was assessed by Annexin V-FITC/Propidium iodide staining and flow cytometry. The results are representative of at least 3 independent experiments, and the error bars signify standard deviations (±SDs). *p<0.01 and **p<0.001 compared with the combination of CX-4945 and gefitinib or erlotinib.