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. 2017 May 23;18(7):1199–1212. doi: 10.15252/embr.201743897

Figure 3. Rhythmic clock gene expression emerges in (matured) cardiac cultures.

Figure 3

  1. BMAL1, PER2, and CLOCK expression levels at D0, D15, and D30 during directed cardiac differentiation as determined using qRT–PCR. Data are represented as mean ± s.e.m. of three independent replicates. Significant expression differences were tested by one‐way ANOVA, followed by a Bonferroni post hoc test (ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.0005).
  2. qRT–PCR analysis of BMAL1 and PER2 expression over 48 h at a 4‐h interval in cardiac cells at D15. Expression levels in (A) and (B) were normalized to PPIA. Data are represented as mean ± s.e.m. of three independent replicates. Significance of rhythmicity across 48 h was analyzed using the RAIN algorithm and is indicated (ns: not significant, ***P < 0.0005).
  3. Promoter‐based destabilized luciferase (dLuc) reporter assay of the Bmal1 and Per2 promoter in synchronized cardiac cells at D15. Values are relative to T0. Measurements were performed using a LumiCycle32.
  4. Similar analysis as in (C) for D30.
  5. Detrended Bmal1‐dLuc and Per2‐dLuc luciferase signal measured in (D).
  6. Detrended Bmal1‐dLuc and Per2‐dLuc bioluminescence in NKX2‐5‐eGFP+ sorted and synchronized human ES cell‐derived cardiomyocytes at D30.
  7. Single‐cell analysis of Per2‐dLuc bioluminescence in sorted eGFP‐positive and synchronized D30 human ES cell‐derived cardiomyocytes.
  8. Representative Per2‐dLuc signal in five single D30 human ES cell‐derived cardiomyocytes over the course of 48 h.
Data information: Measurements in (F–H) were performed with a LV200 microscope.