Fig. 6: GJA1 protected myocardial cells by decreasing cell apoptosis, enhancing cell viability & ATP production rate & oxidative phosphorylation, protecting mitochondrial membrane potential & activating mitophagy. | Cell Death Discovery

Fig. 6: GJA1 protected myocardial cells by decreasing cell apoptosis, enhancing cell viability & ATP production rate & oxidative phosphorylation, protecting mitochondrial membrane potential & activating mitophagy.

From: MiR-130a-3p regulates FUNDC1-mediated mitophagy by targeting GJA1 in myocardial ischemia/reperfusion injury

Fig. 6

A, B Apoptosis in H9c2 cells was measured by Flow Cytometry under normoxia or HR conditions with GJA1 OE. n = 3 in each group. C Cell viability was determined by using a cell counting kit-8 under normoxia or HR conditions with GJA1 OE and absorbance was measured at 450 nm. n = 16 in each group. Relative cell activity (%) = [(As-Ab)/(Ac-Ab)] ×100%. As: absorbance of the experimental group; Ab: absorbance of the blank group; Ac: absorbance of the Ctrl NC group. APC: allophycocyanin; FITC: Fluorescein Isothiocyanate. D Total ATP production rates were measured by Agilent Seahorse analyzer under normoxia or HR conditions. The ECAR and OCR were measured under basal conditions. By obtaining these data under basal conditions and after serial addition of Rotenone/antimycin A, total cellular ATP Production Rates could be measured in real-time. n = 10 in each group. The results of seahorse tests were normalized to the value per 5000 cells. E Mitochondrial respiration was measured including ATP production, maximal respiration, Proton leak, spare respiratory capacity, and basal respiration by Agilent Seahorse analyzer under normoxia or HR conditions. n = 10 in each group. The results of seahorse tests were normalized to the value per 5000 cells. FCCP: Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone. F–G MMP was observed with JC-1 staining. Nuclei were stained with DAPI. The cells were observed by laser confocal microscopy. Fluorescence intensity was measured by ImageJ software. Scale bars, 50 μm and 10 μm in magnified. n = 3 in each group. H The co-localization experiments to observe mitophagy activity. Cells were stained with mito-tracker (green) and lyso-tracker (red). Scale bars, 50 μm and 10 μm in magnified. I The quantitative analysis of the co-localization of lysosome and mitochondria was assessed by the Pearson’s correlation coefficient. J The levels of FUNDC1-mediated mitophagy-associated proteins were assessed by Western blot. n = 3 in each group. Data were expressed as mean ± SD; Statistical significance by two-tailed Student’s t-test, *P < 0.05, **P < 0.01 vs. Ctrl NC; #P < 0.05, ##P < 0.01 vs. HR NC.

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