Figure 5. Blocking MitoJNK signaling prevented stress-related mitochondrial dysfunction and cell death.

(A, B) Interfering with JNK/Sab interaction prevented mitochondrial superoxide generation in anisomycin-stressed HeLa cells. Cells were stressed for 30 minutes with 25µM anisomycin, and then stained with mitochondrial superoxide sensitive dye, MitoSOX Red. Following 10 minutes of staining/stress, the cells were washed, placed in visualizing solution, inspected by microscopy for staining, and then MitoSOX fluorescence was quantitated using a 96-well plated reader. Thirty minutes prior to anisomycin addition cells were either pretreated with 10µM Tat-Scramble, 1µM TI-JIP, or 10µM Tat-Sab_KIM1, A, or cells were transfected with 50nM siRNAs selective for JNK or Sab for 71 hours prior to anisomycin stress, B. (C, D) Preventing JNK mitochondrial translocation decreased dissipation of mitochondrial membrane potential. Cells were stressed with 25µM anisomycin for 45 minutes prior to staining with the mitochondria membrane sensitive dye, JC-1. Cells were stained as indicated with JC-1, and JC-1 staining was confirmed by microscopy prior to quantitation of JC-1 monomer fluorescence by a 96-well plate reader. Cells were either preincubated with 10µM Tat-Scramble, 1µM TI-JIP, or 10µM Tat-Sab_KIM1, C, or exposed to 50nM siRNAs selective for JNK or Sab for 71 hours prior to anisomycin stress, D. (E, F) Inhibition of MitoJNK signaling prevented release of cytochrome c from the mitochondria of anisomycin-treated HeLa cells. Thirty minutes prior to anisomycin addition cells were either pretreated with 10µM Tat-Scramble, 1µM TI-JIP, or 10µM Tat-Sab_KIM1, E, or cells were transfected with 50nM siRNAs selective for JNK or Sab for 71 hours prior to anisomycin stress, F. Cytochrome c levels in the cytosol were quantitated using a cytochrome c ELISA following 4 hours of stress with 25µM anisomycin. (G, H) Disrupting MitoJNK signaling prevented anisomycin-induced cell death in HeLa cells. Cells were either pretreated with 10µM Tat-Scramble, 1µM TI-JIP, or 10µM Tat-Sab_KIM1 for 30 minutes prior to 25µM anisomycin exposure, G, or cells were transfected with 50nM siRNAs selective for JNK or Sab for 71 hours prior to anisomycin stress, H. Cell viability was measured following 4 hours of anisomycin-induced stress or exposure to DMSO. The presence of *s indicate that the conditions connected by lines are statistically significant with a p-value of less than 0.05.