Figure 1.
Figure 1. HNE-induced ROS generation in mitochondria and its role in TF decryption. (A) THP-1 cells were labeled with the mitochondrial staining dye MitoTracker (200 nM) for 30 minutes and then loaded with 5 µM H2DCFDA for 5 minutes at 37°C. Cells were then washed and treated with 20 µM HNE for 20 min at 37°C. After HNE treatment, cells were washed and analyzed for their fluorescence by confocal microscopy. (B) THP-1 cells were treated with specific electron transport chain inhibitors for 1 hour and then loaded with 5 µM H2DCFDA for 5 minutes at 37°C. Cells were treated with HNE for 20 minutes, and ROS generation was analyzed by the fluorescence of H2DCFDA that results from its oxidation by ROS by confocal microscopy. (C) ROS generation was quantified by measuring the fluorescence intensity of the cells as described in “Materials and methods.” The inhibitors used were 2.5 µM rotenone (Rot), 0.5 mM 2-thenoyltrifluoroacetone (THF), 10 µM antimycin (Anti), 10 mM sodium azide (Azide), and 5 µM oligomycin (Oligo). (D-E) THP-1 cells were treated with electron transport chain inhibitors for 1 hour in concentrations indicated in Figure 1B and then 20 µM HNE for 4 hours. Following HNE treatment, cell surface TF (D) and prothrombinase (E) activity was analyzed. (F) THP-1 cells were treated with electron transport chain inhibitors and HNE as described in panels D-E, and PS exposure on the cell surface was analyzed by labeling the fixed cells with AF488-annexin V. 4′-6-Diamidino-2-phenylindole was used to stain nuclei. (G) Quantification of annexin V bound to THP-1 cells (from the fluorescence staining shown in panel F) treated with HNE with or without electron transport chain inhibitors. (H) To investigate the effect of electron transport chain inhibitors on HNE-induced p38 MAPK activation, THP-1 cells were treated with the inhibitors as described above and then treated with HNE for 15 minutes. Cell lysates were subjected to western blot analysis to probe for p38 MAPK phosphorylation and total MAPK. Intensities of phosphorylated and total p38 MAPK bands on the blots were quantified to obtain fold increase in p38 MAPK phosphorylation. *P < .05; **P < .01; ***P < .001; ns, not significant (compared with the values obtained in their respective controls or as indicated in the figures). Images in panels A-B,F were obtained at 63× magnification (oil immersion). Con, control (no treatments); CV, control vehicle; DIC, differential interference contrast; Mito, mitochondria.

HNE-induced ROS generation in mitochondria and its role in TF decryption. (A) THP-1 cells were labeled with the mitochondrial staining dye MitoTracker (200 nM) for 30 minutes and then loaded with 5 µM H2DCFDA for 5 minutes at 37°C. Cells were then washed and treated with 20 µM HNE for 20 min at 37°C. After HNE treatment, cells were washed and analyzed for their fluorescence by confocal microscopy. (B) THP-1 cells were treated with specific electron transport chain inhibitors for 1 hour and then loaded with 5 µM H2DCFDA for 5 minutes at 37°C. Cells were treated with HNE for 20 minutes, and ROS generation was analyzed by the fluorescence of H2DCFDA that results from its oxidation by ROS by confocal microscopy. (C) ROS generation was quantified by measuring the fluorescence intensity of the cells as described in “Materials and methods.” The inhibitors used were 2.5 µM rotenone (Rot), 0.5 mM 2-thenoyltrifluoroacetone (THF), 10 µM antimycin (Anti), 10 mM sodium azide (Azide), and 5 µM oligomycin (Oligo). (D-E) THP-1 cells were treated with electron transport chain inhibitors for 1 hour in concentrations indicated in Figure 1B and then 20 µM HNE for 4 hours. Following HNE treatment, cell surface TF (D) and prothrombinase (E) activity was analyzed. (F) THP-1 cells were treated with electron transport chain inhibitors and HNE as described in panels D-E, and PS exposure on the cell surface was analyzed by labeling the fixed cells with AF488-annexin V. 4′-6-Diamidino-2-phenylindole was used to stain nuclei. (G) Quantification of annexin V bound to THP-1 cells (from the fluorescence staining shown in panel F) treated with HNE with or without electron transport chain inhibitors. (H) To investigate the effect of electron transport chain inhibitors on HNE-induced p38 MAPK activation, THP-1 cells were treated with the inhibitors as described above and then treated with HNE for 15 minutes. Cell lysates were subjected to western blot analysis to probe for p38 MAPK phosphorylation and total MAPK. Intensities of phosphorylated and total p38 MAPK bands on the blots were quantified to obtain fold increase in p38 MAPK phosphorylation. *P < .05; **P < .01; ***P < .001; ns, not significant (compared with the values obtained in their respective controls or as indicated in the figures). Images in panels A-B,F were obtained at 63× magnification (oil immersion). Con, control (no treatments); CV, control vehicle; DIC, differential interference contrast; Mito, mitochondria.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal