134 Chapter 6 Figure 2 A B MM iPSC-CM ACTN1 cTnT DAPI C RPMI-Lac/L MM 40 50 60 70 80 90 100 ACTN1+ cells (%) ** D MM 0 1 2 3 4 5 6 7 8 KI67 + cells (%) *** E MM Starvation Calcein AM EthD1 F H I 105 104 103 0 50K 100K FSC-A 24h 21% O2 150K 200K 250K 102 101 0 24h 1% O2 105 104 103 102 101 0 0 50K 100K FSC-A 150K 200K 250K 0h 4h 16h 24h 0 1 2 3 4 *** *** Viable cells (%) Ratio EthD1/Calcein AM 0 20 40 60 80 Lactate dehydrogenase (U/L) * G 0h 4h 24h Live CMs Live CMs Triton X-100 Cell death (488 nm) 0 20 40 60 80 100 *** RPMI-Lac/L 21% O2 24h 1% O2 4h 1% O2 16h 1% O2 24h 1% O2 1% O2 21% O2 1% O2 Figure 2. Hypoxia induces cell death of MM iPSC-CMs A. Schematic representation of experimental setup. B, C. ACTN1 and cTnT expression of MM iPSC-CMs by immunofluorescence microscopy (B) and flow cytometric analysis of ACTN1 expression (C) (n=8, 2 cell lines, 2 biological replicates, 2 technical replicates). D. KI67 expression of MM iPSC-CMs compared to non-MM iPSC-CMs (n=20, 2 cell lines, 5 biological replicates, 2 technical replicates). E, F. EthD1/Calcein AM staining (LIVE/DEAD assay) of MM iPSC-CMs exposed to 21% O2 for 24 hours or 1% O2 for 4, 8, 16, or 24 hours and quantification (F) (n=15, 2 cell lines, 4 biological replicates, 1-2 technical replicates). G. Lactate dehydrogenase media levels of day 40 matured iPSC-CMs exposed to 1% O2 (n=5, 2 cell lines, 2 biological replicates, 1-2 technical replicates). H, I. Flow cytometry analysis of MM iPSC-CMs viability after 24 hours exposure to 21% O2 or 1% O2 and quantification (I) (n=7, 2 cell lines, 3 biological replicates, 1-2 technical replicates). Data was analysed using one-way ANOVA and Dunnett multiple comparison. *P<0,05; ** P<0,01; ***P<0,001. Scale bar: 200 μm. Data are shown as mean ± SEM.
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