Mitogen deprivation triggers both autophagy and apoptosis.

<p>a-e) GSCs were deprived from mitogens for 1 to 3 days. GSCs growing in mitogen-supplemented medium were use as a control (C). a) Electron microscopy analysis of mitogen-starved GSC#1. <i>Left panels</i>. Cells presented morphological signs of apoptosis, including cytoplasmic blebbing, nuclear fragmentation and chromatin condensation. Scale bars: 2 μm. <i>Right panels</i>. Different stages of autophagy can be seen, such as pre-autophagosome and early autophagosome (upper panels) and late autophagosomes (autolysosomes/amphisomes, lower panels). Scale bars: 50 nm. b) Western blot analysis was conducted using the indicated antibodies in GSC#1. ** non-processed form; * processed form. c-e) LC3B puncta, Atg12 aggregates and mitochondrial cytochrome c release were analyzed by confocal microscopy. Scale bars: 5 μm (c,d) and 2.5 μm (e). f) GSC#1 were pre-treated with vehicle (DMSO) or QVD (10 μM) for 45 min and mitogen-deprived for 3 days in the absence or presence of the drug. Flow cytometry analysis (10.000 events) of Annexin V-FITC/PI staining was used to measure cell death in GSC#1. Number of cells either viable (Annexin V/PI –/–) or in early (Annexin V/PI +/–), late apoptosis (Annexin V/PI +/+) and necrotic (Annexin V/PI –/+) phases was expressed as percentage of total population. g) GSC#1 were transfected with siRNA against Beclin-1 or a control siRNA (siC) for 48 hours. Cells were mitogen-deprived as indicated and collected 5 days post-transfection. Beclin-1 protein levels were analyzed by western-blot and cell death was examined as in f). h) GSC#1 were treated with vehicle (DMSO) or Chloroquine (CQ) (25 μM, XX min) in mitogen-supplemented medium (C) and in mitogen-deprived conditions for 3 days (3d). Flow cytometry analysis (10.000 events) of Annexin V-FITC/PI staining was used to measure cell death in GSC#1 as in f). Graph represents mean+s.d. of three independent experiments. Each panel is representative of three independent experiments.