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. 2014 Jun 5;5(6):e1273. doi: 10.1038/cddis.2014.198

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Copyright © 2014 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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Deletion of FADD affects pre-TCR-mediated apoptosis and proliferation. (a) Cryosections of thymi were stained with TUNEL kit and analyzed by immunofluorescence microscopy to detect apoptosis in the thymus of Lck-FADD mice and littermate control. Scale bar: 100 μm. (b) Histogram of annexin V staining in gated DN3, DN4 and DP thymocytes from 6- to 8-week-old Lck-FADD mice and littermate control. n=4 for each group. (c) Cryosections of thymi were stained for cleaved-caspase-3 expression and analyzed by immunofluorescence microscopy to detect caspase-3 activation in the thymus of Lck-FADD mice and littermate control. Scale bar: 100 μm. (d) Western blot analysis of caspase-8 (upper panel) and caspase-9 (middle panel) levels in thymocytes obtained from Lck-FADD mice and littermate control. The α-tubulin expression served as a loading control (bottom panel). Data are representative of three independent experiments. (e) Flow cytometry of JC-1 cationic dye staining of Lck-FADD mice and littermate control to investigate the mitochondrial membrane potential in the absence of FADD. Potential-dependent accumulation of JC-1 in mitochondria is indicated by a shift in fluorescence emission from green (FL-1) to red (FL-2); numbers beside outlined areas indicate the percentage of cells with decreased ΔΨm in the gated area. Shown are representative data from four independent experiments. (f) Analysis of DN3, DN4 and DP cell proliferation from 6- to 8-week-old Lck-FADD mice and littermate control by EdU incorporation. Shown are representative data from three independent experiments. Error bars reflect S.E.M.