Figure 2. Autophagy is not involved in ETO-induced PCD.

A. Western blots characterizing the expression patterns of proteins involved in autophagy in mESCs prior to and after ETO treatment and recovery. Bafilomycin A1 (BA1) served as a control and inhibits autophagy while allowing for the accumulation of autophagic intermediates. B. Western blot showing level of knockdown of Atg5, Beclin1, or LC3β, after transient transfection of the indicated siRNAs. Cells transfected with siRNAs express GFP, which was probed for to demonstrate equal levels of transfection compared to control vector. C. ESCs were transfected with siRNAs. Twenty four hours posttransfection, cells were treated as indicated and flow cytometry was performed to measure plasma membrane integrity. Cells were first sorted for GFP positive cells prior to analysis for levels of death. Error bars represent the SEM. At least 6 trials are displayed per group. D. ESCs were treated with Spautin 1 or BA1 during ETO treatment and added back during the recovery period. Cells were subjected to flow cytometry to measure the loss of membrane permeability. Error bars represent the SEM. F. ESCs were treated with either NaCl or LiCl₂ during and after ETO treatment and subjected to flow cytometry to analyze loss of plasma membrane integrity. F. Western blot of ESCs treated with NaCl or LiCl₂ probed for LC3. To demonstrate autophagy is increased, BA1 was added to cells for 6 hours at a concentration of 1nM. Two exposures are provided. Actin served as a loading control. ^§p<0.07;*p<0.05; ^#p<0.01.