Figure 4
Blockade of autophagy enhances the anticancer effect of ESI in lung cancer cells. (a) H1299 cells overexpressing mCherry-LC3 were treated with 1.6 μM ESI in presence or absence of 2 mM 3-MA for 24 h, and the cells with mCherry-LC3 punctate dots were examined. Positive signals were defined if the cell have five or more mCherry-LC3 dots in the cytoplasm. (b) The number of mCherry-LC3 dots per cell and (c) the percentage of the cells with mCherry-LC3 dots were counted under florescence microscope. (d and e) Both H1299 and A549 cells were pretreated with 2 mM 3-MA, an inhibitor of autophagy, for 1 h and then exposed to ESI (1.6 μM for H1299, 3.2 μM for A549) for another 24 h. (d) Western blot analysis of Beclin1, ATG3 and LC3 expression levels in the H1299 and A549 cells. Actin was used as loading control. (e) The cell viability was determined by WST-1 assay. (f) H1299 and A549 cells were treated with ESI alone or together with 2 mM 3-MA for 24 h, and then apoptotic cells were detected with the Annexin V-APC/7-AAD kit and analyzed by flow cytometry. All data were representative of three independent experiments. Bars, S.E.M.; *P<0.05, **P<0.01, ***P<0.001
