Figure 3

A) Wild type α-sarcin (wt) and the R121Q and H137Q mutants induced cytotoxicity in HeLa cells in a dose-dependent manner. HeLa cells were transfected with increasing amounts of toxin cDNA and assayed for viability, relative to a pcDNA3 control, at 48 hours post-transfection. B) Inhibition of GFP translation by α-sarcin wt, R121Q, or H137Q mutants determined by western blotting. C) Inhibition of firefly luciferase expression relative to pcDNA3/luciferase co-transfected cells, as determined by luminescence analysis of cell lysates. D) α-Sarcin wt induces JNK activation whereas the catalytic mutants do not. pJNK Western blot (arrow indicates position of JNK-2) HeLa cell lysates transfected with α-sarcin wt, R121Q mutant, H137Q mutant, 3 × Flag-sarcin or treated with vehicle (DMSO) or anisomycin for 20 minutes. Anisomycin is a potent agonist of JNK and is included as a positive control. (Bottom) For a loading control the above blot was stripped and reprobed with anti-JNK antibody. E) Quantification of JNK activation relative to pcDNA transfected controls as determined using a Li-Cor infrared imager. Charted data represents the compiled results of 3 independent experiments, each performed in triplicate, +/- standard deviation. F) Relative viability of Cos 7 cells transfected with pcDNA3-α-sarcin (wt) or R127Q, or H137Q mutants.