Mitogen deprivation reduced glioblastoma stem-like cell survival.

<p>a-h) GSCs were deprived from mitogens for 1 to 3 days. GSCs growing in mitogen-supplemented medium were used as a control (C). a) The number of secondary neurospheres per field of view (NS/FOV) was counted after 3 days of deprivation in GSCs #1-4. b) Sox2 levels (green) were analyzed by confocal in GSCs #1-4. Nuclei were counterstained with DAPI (blue). Scale bar: 15 μm. c-d) Expression of Sox2 and Nestin as well as GADPH as a control, was evaluated by RT-PCR, while GFAP expression was analyzed by confocal analysis. Differentiation of GSC#4 was induced as described in methods. e) PI incorporation was measured by flow cytometry (10.000 events) in GSCs #1–14 and the percentage of cell death was estimated. Graph represents mean+s.d. of three independent experiments. Student’s t-test: ^***<i>P</i><0.001, ^**<i>P</i><0.01. f) Flow cytometry analysis (10.000 events) of Annexin V-FITC/Propidium Iodide (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) DNA profile was analyzed by flow cytometry with PI staining in GSC#1. Percentage of apoptotic cells was calculated based on sub-G₁ peak. Student’s t-test: ^***<i>P</i><0.001. h) Western blot analysis was conducted using the indicated antibodies in GSC#1. Each panel is representative of three independent experiments.