fig1

Figure 1. Injected STEMIN and YAP5SA mmRNA induced nuclear replication in infarcted adult mouse hearts. (A) Schematic diagram of short-term (24 h) in vivo experiment. A combination of STEMIN and YAP5SA mmRNA was injected in five locations around the infarct in the left ventricle of inbred litter mates (100 days old). (B) Mouse IVIS bioluminescence images 24 h post-injection: (Left) mouse heart injected with 70 µg (50 µL) Luc mmRNA + 32 µL Lipofectamine MessengerMAX; and (Right) non-Luc control mouse heart injected with PBS (50 µL) + 32 µL Lipofectamine MessengerMAX. D-luciferin (150 mg/kg mouse body weight) injected via subcutaneous injection 4 h before light emission analysis to detect luciferase signal using an IVIS BioImager. (C) Images of DAPI and EdU staining of paraformaldehyde fixed left ventricle cardiac sections taken 24 h after five equal injections of STEMIN (50 µg in 38 µL) and YAP5SA (50 µg in 22 µL) with 33 µL Lipofectamine MessengerMAX around the infarct. The control group was injected with PBS (60 µL) and 33 µL Lipofectamine MessengerMAX. EdU (10 μg/g of mouse body weight) was injected via subcutaneous injection at 8 h prior to sacrifice. The intact heart was immediately washed and drained of blood cells, fixed in 4% paraformaldehyde, stored in 70% ethanol, and then embedded into paraffin for histological assessment. Each heart was cross-sectioned and EdU was detected by Click-iT EdU Cell Proliferation Kit (ThermoFisher). Note the overlapping images stained along three needle tracts for DAPI, EdU, Anti-SRF, and Anti-YAP. (D) Merged images of DAPI and EdU staining were first divided into 90 equal optical slices and then integrated density was measured, in which the same threshold was used for all the optical slices.