Supplementary Materials

The PDF file includes:

  • Methods
  • Fig. S1. Approach for determining the ploidy of 15N-thymidine–labeled nuclei.
  • Fig. S2. The two nuclei in binucleated cardiomyocytes have the same 15N-thymidine intensity, demonstrating that they share the same cell cycle history.
  • Fig. S3. A single-cell transcriptional profiling strategy identifies molecular mechanisms of cardiomyocyte binucleation.
  • Fig. S4. Strategy for isolation and characterization of cycling cardiac cells.
  • Fig. S5. Strategy for identifying single mouse cardiomyocytes for single-cell transcriptional analysis.
  • Fig. S6. The expression of Ect2 gene decreases in cycling neonatal mouse cardiomyocytes.
  • Fig. S7. Adenoviral-mediated transduction efficiency of Ect2 in NRVMs is >90%.
  • Fig. S8. Adenoviral-mediated transduction of GFP-Ect2 does not induce apoptosis in cultured NRVMs.
  • Fig. S9. Survival analysis shows that Ect2 gene inactivation in development induces decreased pup viability.
  • Fig. S10. Inactivation of Ect2 gene in development does not induce apoptosis.
  • Fig. S11. The WT Ect2 promoter was modified to test the effect of the putative TEAD1/2-binding sites on the Ect2 promoter activity.
  • Fig. S12. Knockdown of Ect2 using siRNA reduces Ect2 mRNA and protein and induces cytokinesis failure and binucleation in cardiomyocytes.
  • Fig. S13. Approaches for quantification of BrdU+ cardiomyocytes in vivo.
  • Fig. S14. Altering β-AR signaling does not affect the heart weight.
  • Fig. S15. The optical dissector operates optimally at 3-μm distance between lookup and counting frames.
  • Fig. S16. Validation of BrdU assay specificity in cultured neonatal cardiomyocytes.
  • Fig. S17. Representative immunostained cardiomyocytes isolated from cultured human myocardium.
  • Legend for table S1
  • Table S2. Cell cycle genes for Fig. 8A.
  • Table S3. Quantification of numeric data.
  • Table S4. Antibody manufacturers and dilutions.
  • Table S5. Image acquisition hardware and settings.
  • Table S6. PCR primers and 5′-3′ oligonucleotides.
  • Table S7. Animal strains used in this study.
  • Table S8. Clinical information corresponding to human samples.
  • Legends for movies S1 to S8
  • References (7075)

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Other Supplementary Material for this manuscript includes the following:

  • Table S1 (Microsoft Excel format). List of differentially expressed genes encoding 61 different Dbl homology family Rho-GEFs between E14.5 and P5 cycling and noncycling mouse cardiomyocytes.
  • Movie S1 (.mov format). Live cell imaging in NRVMs shows that cleavage furrow regression precedes formation of binucleation.
  • Movie S2 (.mov format). Live cell imaging of a neonatal cardiomyocyte undergoing division.
  • Movie S3 (.mov format). Live cell imaging of a neonatal cardiomyocyte undergoing cytokinesis failure.
  • Movie S4 (.mov format). Live cell imaging shows appropriate and dynamic localization of GFP-Ect2 during the cell cycle.
  • Movie S5 (.mov format). Heart function of newborn (P0) WT mice.
  • Movie S6 (.mov format). Heart function of newborn (P0) mouse pup with Ect2 knockout.
  • Movie S7 (.mov format). Ect2 inactivation induces heart failure and death of newborn mice.
  • Movie S8 (.mov format). Heart function of the left anterior descending coronary artery (LAD) ligation–injured adult mouse.