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. Author manuscript; available in PMC: 2013 Jun 21.
Published in final edited form as: Nat Chem Biol. 2012 Oct;8(10):831–838. doi: 10.1038/nchembio.1059

Figure 1.

Figure 1

Exogenous C18-Pyr-Cer (C18PC) induces autophagic cell death. (a) C18-Pyr-Cer. (b) Effects of C18-Pyr-Cer on the lipidation of LC3B (LC3B-II) were examined by western blot in the absence or presence of a pan caspase inhibitor Z-VAD compared to vehicle-treated controls (lanes 1-2 and 3-4, respectively). Full blots can be found in Supplementary Figure 13. (c) Formation of double-membrane autophagosomal vesicles in the absence or presence of C18-Pyr-Cer was visualized by TEM (left and right panels, respectively). Higher magnification of TEM visualization is shown in lower panels. Scale bars represent 10 microns (top) and 500 nm (bottom). (d) Effects of siRNA-mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18-Pyr-Cer were determined compared to controls transfected with Scr siRNAs using trypan blue exclusion assay. (e) Roles of C18-Pyr-Cer in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wt (ATG5+/+) versus ATG5-/- k/o mice with C18-Pyr-Cer. (f) Effects of C18-Pyr-Cer on caspase-dependent or –independent cell death were determined in MEFs isolated from Bax-/-/Bak-/- (dko) or caspase3-/-/7-/- (dko) mice compared to cells isolated from wt (Bax+/+/Bak+/+) or caspase3+/-/7+/- mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P <0.05).