Figure 10. JCTH-4 directly causes mitochondrial ROS production and release of apoptogenic factors independent of caspases.

(A) Saos-2 and (B) U-2 OS isolated mitochondria were treated directly with JCTH-4, CC, PQ, and solvent control (Me₂SO), and incubated with Amplex Red and horseradish peroxidase for 2 hours. Subsequently, fluorescence readings were taken at Ex. 560 nm and Em. 590 nm and expressed as relative fluorescence units (RFU). Statistics were performed using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean ± SD of quadruplicates of 1 independent experiment. *p<0.05, **p<0.01, ***p<0.001 versus solvent control (Me₂SO); †p<0.01 versus 0.25 µM JCTH-4; @p<0.01 versus 0.5 µM JCTH-4; #p<0.01 versus 5 µM CC. Isolated mitochondria samples treated directly with JCTH-4, CC, and solvent control (Me₂SO) for 2 hours were also centrifuged, producing mitochondrial pellets and post mitochondrial supernatants which were examined for retention and release of apoptogenic factors respectively via western blot analyses; (C) Retention of AIF and release of EndoG by U-2 OS cell mitochondria and (D) release of AIF by Saos-2 cell mitochondria was monitored. Mitochondrial pellets were probed for SDHA to serve as loading controls. Densitometric analyses were performed using ImageJ software and statistics were calculated using GraphPad Prism version 5.0. Image is representative of 3 independent experiments demonstrating similar trends. Values are expressed as mean ± SD of triplicates of one independent experiment. *p<0.01, **p<0.001 versus solvent control (Me₂SO); †p<0.01 versus 0.25 µM JCTH-4; #p<0.01 versus 5 µM CC. (E) Saos-2 cells were treated with broad spectrum caspase inhibitor Z-VAD-FMK with and without JCTH-4 for 72 hours. WST-1 reagent was used to quantify cell viability. Absorbance was read at 450 nm and expressed as a percent of solvent control (Me₂SO). Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *p<0.001 versus solvent control (Me₂SO); ns = not significant.