1. Fig. 4 Analysis of ISO response in vitro in neonatal cardiomyocytes.
Analysis of ISO response in vitro in neonatal cardiomyocytes. (A) Mitochondrial respirometry analysis of neonatal cardiomyocytes upon different treatments measured by Seahorse and maximum respiration rate (MRR) quantification. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FBS, fetal bovine serum; NAC, N-acetyl-l-cysteine; OCR, oxygen consumption rate. (B) 2′,7′-Dichlorofluorescein diacetate (H2-DCFDA)–mediated ROS quantification in neonatal cardiomyocytes upon different treatments. (C) Fura-2-acetoxymethyl ester (Fura-2 AM)–mediated intramitochondrial calcium quantification in neonatal cardiomyocytes upon different treatments. (D) Tetramethylrhodamine methyl ester perchlorate (TMRM)–mediated mitochondrial membrane potential determination in neonatal cardiomyocytes upon different treatments. (E) Analysis of OPA1 processing by Western blot. (F) Analysis of MCU expression by Western blot of mitochondrial lysate. SDHA and core2 are used as loading controls. (G) Kurtosis analysis as indicator of calcium distribution in the cardiomyocytes at the indicated treatments. Basal and final treatments correspond to time 0 and time 9 min, respectively. (H) Time-lapse images (40×) at the indicated time showing calcium (Fura-2 AM) and mitochondrial (MitoTracker Deep Red) distribution under different treatments. Scale bars, 20 μM. Data in upper and middle panels of (A) are given as means ± SEM and in the rest of graphics as scatter dot plots, and lines are means ± SD. Differences assessed by two-way ANOVA and Sidak’s multiple comparison test (A) or one-way ANOVA and Tukey’s multiple comparison test (B to F). N as described in table S1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <
Rebeca Acin-Perez et al., Sci Transl Med 2018;10:eaan4935
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