fig5

Figure 5. SRF153(A3) mutant and YAP5SA mutant mmRNA transduce NRVM proliferation. (A) Schematic protocol of transfected synthetic SRF153(A3) and YAP5SA mmRNA and/or in combination all with Lipofectamine MessengerMAX into NRVM to test for cell replication. (B) Optimal doses of synthetic mmRNA with Lipofectamine MessengerMAX were transfected in NRVM for 24 h and then pulsed for an additional 8 h with alpha-EdU. Controls did not receive mmRNA. NRVM were nuclear blue stained with DAPI. Alpha-EdU incorporated into the DNA of replicating myocytes was stained red with a Click-It kit. Anti-TNNT was used to stain organized contractile fibrils (green). White boxes mark replicated nuclei. (C) An increased number of myocytes is shown by the significant increase in DAPI stain nuclei for transfected SRF153(A3) and YAP5SA mmRNA as well as in combination. (D) EdU incorporation plotted as a ratio to DAPI 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 and **P < .005. (E) The ratio of EdU/DAPI-labeled cardiomyocytes was quantitated at three different time points, each with a final 8 h EdU pulse. (F) The first 24 h revealed the highest ratio of alpha-EdU/DAPI-labeled cardiomyocytes for the combination of mmRNA. The following time points showed reduced alpha-EdU/DAPI ratios, indicating declining EdU incorporation per nucleus. The number of cells ranged from 700 to 2800 in triplicates.