mTOR Inhibition by Everolimus in Childhood Acute Lymphoblastic Leukemia Induces Caspase-Independent Cell Death
Figure 3
Everolimus-Induced Autophagy is Not Required for Cell Death.
(A–G) NALM6 cells were treated with vehicle or 16 µM everolimus overnight and ultrastructure examined by electron microscopy. Fine arrows indicate autophagic vacuoles containing degrading organelles. Thick arrows indicate stretches of double membrane walling off regions of cytoplasm. Scale bars indicate 100 nm. (H) Cell lysates prepared from NALM6 cells cultured for the indicated time periods with the specified concentration of everolimus were analyzed for LC3-I and LC3-II by Western blotting and sequential blotting for β-actin used as a loading control. (I) The viability of NALM6 cells cultured for 24 h with the indicated concentration of everolimus was assessed with and without the addition of 3MA. The mean ± SD of 3 independent experiments is shown and analyzed using a paired t-test. (J) Analysis of LC3 by Western blotting following a 24 h incubation of NALM6 cells with indicated concentration of everolimus with or without that addition of 3MA. The ratio of LC3-II to LC3-I is indicated below the blot.