136 Chapter 6 Figure 3 B 5% O2 24h 5% O2 21% O2 4h 1% O2 1% O2 C 4h 24h 24h D 24h TUNEL DAPI 24h E Bright field Bright field 0 4 24 0 10 20 30 40 50 Hours TUNEL+ CMs (%) 21% O2 5% O2 1% O2 *** * 24h ACTN1 cTnT DAPI A F 21% O2 1% O2 1% O2 4h 24h 24h 4h 4h 24h 24h 5% O2 21% O2 1% O2 1% O2 5% O2 5% O2 1% O2 0 4 24 Hours 0 1 2 3 Glucose (mmol/L) * P=0,051 Figure 3. Mild hypoxia is sufficient to induce cell death in MM iPSC-CMs Schematic representation of experimental set-up. B. Bright-field images of MM iPSC-CMs exposed to indicated hypoxia conditions. C. Microscopic images of ACTN1 and cTnT immunostainings. Square indicates area of close-up. D. Microscopic images of TUNEL staining of MM iPSC-CMs. E. Quantification of TUNEL staining. F-H. Glucose (F) and lactate (G) concentrations in media after 4 to 24 hours incubation of MM iPSC-CMs at indicated hypoxia conditions. E: 3 technical replicates from 6 biological replicates (cardiac differentiations) (n=28); F, G: 2 technical replicates from 3 biological replicates (cardiac differentiations) (n=5). Data were 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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