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Figure 6. Transfected SRF153(A3) and YAP5SA mmRNA induced nuclear replication and the appearance of cleavage furrows in adult rat cardiomyocytes. (A) Schematic diagram of SRF153(A3) and YAP5SA mmRNA mutants and/or in combination transfected into three-month-old rat primary cardiac myocytes isolated by the Langendorff system. Alpha-EdU was pulsed in cultures to stain DNA synthesis in nuclei within 24 h after mmRNA transfection. (B) Alpha-EdU pulse assay of adult CM treated with wild-type SRF and YAP. Control myocytes were treated with Lipofectamine MessengerMAX. Wild-type SRF and the YAP-treated group barely displayed the alpha-EdU signal. (C) Alpha-EdU pulse assay of adult CM treated with SRF153(A3) and YAP5SA. (D) Notably, more than 90% of cardiomyocytes had alpha-EdU/nuclei positive signals, which was significantly increased in all mmRNA treatment groups compared to the control group, as determined by two-tailed statistical analysis, in which *P < 0.05. (E) Adult CM treated SRF153(A3) and YAP5SA were stained with DAPI, anti-TNNT, and anti-DIAPH3. Immuno-fluorescence microscopy showed DIAPH3 localized in multiple regions between and surrounding dividing nuclei, as shown by white arrows. (F) TNNT marker in the mRNA-treated adult mouse cardiomyocytes showed reduced TNNT immunofluorescence around the nuclei (marked by dashed circles) in the SRF153(A3) mmRNA and the SRF153(A3) and YAP5SA mmRNA combo treated group. Cell cycle cleavage furrows were marked by white arrows.
