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Translocation of BAX to mitochondria, release of cytochrome C, and activation of caspase-8. ( A ) Quantitative sandwich ELISA immunoassay showed a significant increase in Bcl-2-associated X (BAX) within the mitochondrial fraction of hypoxic cardiomyocytes (CMs). Moreover, hypoxic CMs preconditioned with H-sEVs displayed significantly higher (+51.6%) mitochondrial levels of BAX compared with hypoxic control cells. ( B ) Inversely, the cytosolic levels of BAX were significantly lowered after 18 h of hypoxia. There were no significant differences in cytosolic BAX levels between the two hypoxic groups. ( C ) Quantitative sandwich ELISA immunoassay showed a significant increase in Cytochrome C within the cytosolic fraction of hypoxic CMs. Interestingly, 24 h preconditioning with H-sEVs significantly increased Cytochrome C translocation (+70%) compared with control cells. ( D ) Inversely, the mitochondrial levels of Cytochrome C were significantly reduced in the hypoxic CMs. In addition, hypoxic CMs preconditioned with H-sEVs displayed significantly lower levels of Cytochrome C (−32.7%) within the mitochondrial fractions compared with hypoxic control cells. ( E ) Caspase-8 activity assay showed that 18 h of hypoxia increase caspase-8 activity in AC16 CMs. Furthermore, preconditioning with H-sEVs significantly increased caspase-8 activity (+110%) compared with control cells. To validate the integrity of the mitochondrial and cytosolic fractions, we performed several quantitative sandwich ELISA immunoassays. The mitochondrial fractions were validated by the absence of β-Actin and the presence of Cytochrome C Oxidase Subunit 4 (COX4), while the cytosolic fractions were validated by the presence of β-Actin and the absence of COX4 . Data was analyzed using one-way ANOVA multiple comparison analysis. Data is expressed as mean of raw values ± SEM ( n = 4). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Journal: International Journal of Molecular Sciences

Article Title: Hypoxic Small Extracellular Vesicle Preconditioning of AC16 Cardiomyocytes Increase Caspase-3 and Caspase-8 Activity During Hypoxia

doi: 10.3390/ijms262412123

Figure Lengend Snippet: Translocation of BAX to mitochondria, release of cytochrome C, and activation of caspase-8. ( A ) Quantitative sandwich ELISA immunoassay showed a significant increase in Bcl-2-associated X (BAX) within the mitochondrial fraction of hypoxic cardiomyocytes (CMs). Moreover, hypoxic CMs preconditioned with H-sEVs displayed significantly higher (+51.6%) mitochondrial levels of BAX compared with hypoxic control cells. ( B ) Inversely, the cytosolic levels of BAX were significantly lowered after 18 h of hypoxia. There were no significant differences in cytosolic BAX levels between the two hypoxic groups. ( C ) Quantitative sandwich ELISA immunoassay showed a significant increase in Cytochrome C within the cytosolic fraction of hypoxic CMs. Interestingly, 24 h preconditioning with H-sEVs significantly increased Cytochrome C translocation (+70%) compared with control cells. ( D ) Inversely, the mitochondrial levels of Cytochrome C were significantly reduced in the hypoxic CMs. In addition, hypoxic CMs preconditioned with H-sEVs displayed significantly lower levels of Cytochrome C (−32.7%) within the mitochondrial fractions compared with hypoxic control cells. ( E ) Caspase-8 activity assay showed that 18 h of hypoxia increase caspase-8 activity in AC16 CMs. Furthermore, preconditioning with H-sEVs significantly increased caspase-8 activity (+110%) compared with control cells. To validate the integrity of the mitochondrial and cytosolic fractions, we performed several quantitative sandwich ELISA immunoassays. The mitochondrial fractions were validated by the absence of β-Actin and the presence of Cytochrome C Oxidase Subunit 4 (COX4), while the cytosolic fractions were validated by the presence of β-Actin and the absence of COX4 . Data was analyzed using one-way ANOVA multiple comparison analysis. Data is expressed as mean of raw values ± SEM ( n = 4). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: BAX antibody-blocking peptide , ELISA Detection , N/A , N/A , Novus Biologicals (Centennial, CO, USA) , NBP1-88682PEP , N/A.

Techniques: Translocation Assay, Activation Assay, Sandwich ELISA, Control, Activity Assay, Comparison