Fig. 6.

Stat3–Grim-19 directly induces mitochondrial damage that is dependent on mitoneet activation. (A) Hepatocytes were transfected with siRNA targeting mitoneet or a non-targeting control siRNA. After 48 hours, hepatocytes were loaded with 200 nM TMRM for 30 minutes. The cells were then washed twice with PBS and incubated further for 5 minutes in respiratory buffer containing 20 nM of TMRM. Digitonin at 2.5 µg/ml was then added to permeabilize the plasma membrane. Where indicated, hepatocytes were pre-treated with 10 µM of Ru360 or 10 µM of pioglitazone for 5 minutes before the addition of the Stat3–Grim-19 complex. The recombinant complex of Stat3-Grim-19 was added at a final concentration of 100 µM. TMRM fluorescence was monitored over a 20 minute time course. 5 µM of CCCP was added at the 18 minute time point. The result is the average of three independent experiments. (B) Hepatocytes as in A were loaded with MitoSOX for 30 minutes. Digitonin (2.5 mg/ml final concentration) was then added to permeabilize the plasma membrane. Time-lapse microscopy was conducted over a 20 minute time course. Recombinant Stat3–Grim-19 complex was added at a concentration of 100 µM at the 2 minute time point with MitoSOX fluorescence intensity assessed. Values are the means ± s.d. of three independent experiments. (C) Hepatocytes treated as in A were labeled for 15 minutes with 1 mM RPA in Williams Medium E containing 10 mM HEPES buffer instead of Phenol Red. Where indicated, the cells were pre-treated for 10 minutes with 10 µM of pioglitazone or 10 µM of Ru360. Digitonin (2.5 mg/ml final concentration) was added to permeabilize the plasma membrane. Recombinant Stat3–Grim-19 complex was added at a concentration of 100 µM and fluorescence was monitored. At the end of the time course, 5 mM of FeCl₃-8-hydroxyquinoline (FHQ) was added so as to attain maximal quenching. The result is the average of three independent experiments.