Fig. 3.
TLR2 activation has no impact on mitochondrial ΔΨm and intracellular ATP production. (A, B) Mitochondrial ΔΨm measurements. Mitochondrial ΔΨm was detected with TMRE (A) or JC-1 (B) dye. (A) Peritoneal leukocytes were treated with the indicated concentrations of antimycin A or Pam3Cys for 1 h and analyzed for ΔΨm with flow cytometry. ***P < 0.001 vs. control. The numbers of samples in each group: 0 μg/ml antimycin A, n = 7; 1 μg/ml antimycin A, n = 5; 10 μg/ml antimycin A, n = 3; 20 μg/ml Pam3cys, n = 4. The experiments were performed twice. (B) Cells were treated with antimycin A or TLR ligands as indicated: Pam3Cys, LTA, LPS, poly (I:C) or CpG, all at 20 μg/ml, for 1 h and analyzed for ΔΨm with fluorescence ratio detection. n = 3 in each group. *P < 0.05, ***P < 0.001 vs. control. The experiments were performed twice. (C, D) ATP production in the presence or absence of glucose. (C) Cells were treated with antimycin A, Pam3Cys, or LPS (all at 20 μg/ml) in glucose containing medium for 4 h and analyzed for ATP production with a ATP bioluminescence assay kit. n= 3 in each group. The experiments were performed three times. (D) Cells were treated with antimycin A, Pam3Cys, or LPS (all at 20 μg/ml) in glucose-free medium for 1 h and analyzed for intracellular ATP level, ***P < 0.001 vs. control. Each error bar represents mean ± SEM. The numbers of samples in each group: control, n = 5; antimycin A, n = 5; Pam3cys, n = 5; LPS, n = 5. ΔΨm = membrane potential; ATP = adenosine triphosphate; LPS = lipopolysaccharides; LTA = lipoteichoic acid; MFI = mean fluorescence intensity; TMRE = tetramethylrhodamine ethyl ester perchlorate.

TLR2 activation has no impact on mitochondrial ΔΨm and intracellular ATP production. (A, B) Mitochondrial ΔΨm measurements. Mitochondrial ΔΨm was detected with TMRE (A) or JC-1 (B) dye. (A) Peritoneal leukocytes were treated with the indicated concentrations of antimycin A or Pam3Cys for 1 h and analyzed for ΔΨm with flow cytometry. ***P < 0.001 vs. control. The numbers of samples in each group: 0 μg/ml antimycin A, n = 7; 1 μg/ml antimycin A, n = 5; 10 μg/ml antimycin A, n = 3; 20 μg/ml Pam3cys, n = 4. The experiments were performed twice. (B) Cells were treated with antimycin A or TLR ligands as indicated: Pam3Cys, LTA, LPS, poly (I:C) or CpG, all at 20 μg/ml, for 1 h and analyzed for ΔΨm with fluorescence ratio detection. n = 3 in each group. *P < 0.05, ***P < 0.001 vs. control. The experiments were performed twice. (C, D) ATP production in the presence or absence of glucose. (C) Cells were treated with antimycin A, Pam3Cys, or LPS (all at 20 μg/ml) in glucose containing medium for 4 h and analyzed for ATP production with a ATP bioluminescence assay kit. n= 3 in each group. The experiments were performed three times. (D) Cells were treated with antimycin A, Pam3Cys, or LPS (all at 20 μg/ml) in glucose-free medium for 1 h and analyzed for intracellular ATP level, ***P < 0.001 vs. control. Each error bar represents mean ± SEM. The numbers of samples in each group: control, n = 5; antimycin A, n = 5; Pam3cys, n = 5; LPS, n = 5. ΔΨm = membrane potential; ATP = adenosine triphosphate; LPS = lipopolysaccharides; LTA = lipoteichoic acid; MFI = mean fluorescence intensity; TMRE = tetramethylrhodamine ethyl ester perchlorate.

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