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. 2019 Dec 31;30:101422. doi: 10.1016/j.redox.2019.101422

Fig. 6.

Fig. 6

Downregulation of Raptor expression induces mitochondrial ROS production. (A) Representative confocal images obtained on cells stably transfected with a pDsRed2-mito (red) plasmid labeling mitochondria after treatment with 2 μM ODN for 6 h. The nuclei were stained with DAPI (blue), and the length of the mitochondria was measured using LSM 5 Image Browser. (B–E) Caki cells were treated with 2 μM ODN for the indicated time (B and E) or 6 h (C and D) and analyzed for indicated proteins by western blotting (B). Flow cytometry was used to detect fluorescence intensity for mitochondrial damage (C) and ATP production was measured using an ATP determination kit (D). Flow cytometry was used to detect fluorescence intensity for mitochondrial ROS (E). (F and G) Caki cells were pretreated with different concentrations of Mito-TEMPO and treated with 2 μM ODN and 50 ng/mL TRAIL (F) or 2 μM ODN (G) for 24 h, and apoptosis and protein expression were measured by flow cytometry and western blotting, respectively. (H and I) Caki cells were transfected with pRK5 (Caki/Vec) or pRK5-Myc-Raptor (Caki/Raptor) and treated with 2 μM ODN for 6 h (H) or 24 h (I). Mitochondrial ROS production was analyzed by MitoSOX Red reagent staining and flow cytometry. (J) Representative confocal images obtained on Caki/pDsRed2-mito cells transfected with pRK5 (Vec) or pRK5-Myc-Raptor (Raptor) after treatment with 2 μM ODN for 6 h. The nuclei were stained with DAPI, and the length of the mitochondria was measured using LSM 5 Image Browser. (K) Caki cells were pretreated with different concentrations of Mito-TEMPO or MnTMPyP and treated with 2 μM ODN for 24 h. (L) Caki cells were transfected with pRK5 (Vec) or pRK5-myc-Raptor (Raptor) and treated with 2 μM ODN for 24 h. (M) Caki cells were pretreated with different concentrations of Mito-TEMPO or MnTMPyP and treated with 2 μM ODN for 24 h. Apoptosis and protein expression were measured by flow cytometry and western blotting, respectively. The values in graph (A, D-F, H and J) represent the mean ± SD of three independent experiments. *p < 0.01 compared to the control. #p < 0.01 compared to the combinations of ODN and TRAIL. &p < 0.01 compared to the ODN treatment in Caki/Vec. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)