140 Chapter 6 Figure 5 A B 21% O2 24h Calcein AM EthD1 1% O2 24h 1% O2 24h/Nec-1 C D 0 50 100 150 Mitochondrial Respiration OCR (pMol/min) Spare Respiratory Capacity Maximal Respiration Basal Respiration ATP production Proton Leak Non mitochondrial Respiration E **** **** ** **** **** * **** **** *** *** **** ** **** ** F TOMM20 ACTN1 DAPI G 0 25 50 75 100 TOMM20 intensity/nuclei (%) *** * 1 10 20 30 40 50 60 70 80 90 100 110 0 50 100 150 Time (min) Oligomycin FCCP Antimycin A & Rotenone Mitochondrial Respiration OCR (pMol/min) 21% O2 24h 1% O2 24h 1% O2 24h/Nec-1 0 1 2 3 4 Ratio EthD1/Calcein AM * ** 21% O2 24h 1% O2 24h 1% O2 24h / Nec-1 21% O2 24h 1% O2 24h 1% O2 24h/Nec-1 Nec-1 21% O2 24h 1% O2 24h 1% O2 24h/Nec-1 Figure 5. Preconditioning with Nec-1 protects MM iPSC-CMs from hypoxia A. Schematic representation of experimental set-up. B. EthD1/Calcein AM staining of MM iPSC-CMs exposed to 21% O2 or 1% O2 for 24 hours in the absence or presence of necroptosis inhibitor Nec-1. C. Quantification of B. D-E. Normalized real-time oxygen consumption rate (OCR) of iPSC-CMs MM iPSC-CMs in normoxia (21% O2, 24 hours), long-term ischemia (1% O2, 24 hours), or long-term hypoxia preconditioned with Nec-1 for 24 hours by Seahorse extracellular flux analyser. Cells were treated with oligomycin, FCCP, and antimycin A and rotenone to measure mitochondrial respiration. F. Immunofluorescent staining of sarcomeres (ACTN1, green) and mitochondria (TOMM20, red) of cells used for metabolic profiling. G. Quantification of TOMM20 staining. D-F: 2 cell lines, 2 biological replicates, 1-2 technical replicates (n=5). Scale bars: 200 μm. Data were analysed using two-way ANOVA. *P<0,05; **P<0,01; ***P<0,001; ****P<0,0001. Data are shown as mean ± SEM.
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