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. Author manuscript; available in PMC: 2021 Apr 14.
Published in final edited form as: Circulation. 2020 Feb 11;141(15):1249–1265. doi: 10.1161/CIRCULATIONAHA.119.043067

Figure 4. Pkm2 directly interacts with β-catenin, upregulates its downstream targets (Cyclin D1 and C-Myc) and increases expression of cell-cycle-promoting genes and cell cycle markers in postnatal CMs.

Figure 4.

A&B. 2 days post MI and administration of cmsLuc or cmsPkm2 modRNAs with cmsihCD25, adult CMs were isolated using magnetic beads (please see method in Suppl. Fig. 13). A. qRT-PCR analysis to validate purity of isolated CMs, CM-specific markers and functional cardiac genes (n=3). B. Gene expression comparisons of β-catenin downstream target genes and expression of cell-cycle-promoting genes or cell-cycle inhibitors post cmsLuc or cmsPkm2 modRNA delivery into adult CMs (n=3). C. Co-Immunoprecipitation (Co-IP) and input control to evaluate Pkm2’s direct interaction with β-catenin in P3 rat neonatal CMs post Luc or Pkm2 modRNA transfection (n=2). D. Immunostaining of rat neonatal CM after Luc or Pkm2 modRNA delivery shows co-localized β-catenin and Pkm2 in the CM nucleus. E. Stabilizing β-catenin induced its interaction with TCF/LEF transcription factors in the nucleus, which in turn induced the expression of target genes (e.g cyclin D1. c-Myc) that can be measured by TOPflash (Luciferase reporter with TCF binding DNA sequence). Fopflash has mutant TCF binding DNA sequence (Fopflash), used as control. Quantitative TOPflash or Fopflash following delivery of nGFP (control) or Pkm2 modRNA (n=3). F. Experimental plan for evaluating overexpression of β-catenin alone or with Pkm2 modRNA and studying its effect on the expression of cell cycle genes in P3 neonatal rat CMs. G. Representative images of β-catenin expression 1 day post Luc or β-catenin modRNA transfection in vitro (co-stained α-Actinin and DAPI). H&I. Quantification of cell cycle markers (Ki67, pH3) in neonatal rat CMs 3 days after transfection of Luc, Pkm2 or β-catenin modRNA alone or combined Pkm2 and β-catenin modRNAs (n=3). J. Experimental timeline used to evaluate the role of β-catenin in CMs post Pkm2 modRNA delivery. K. Representative images of β-catenin inhibition by siRNA in CMs after Pkm2 modRNA delivery. L&M. Quantification of cell cycle markers (Ki67, pH3) in neonatal rat CMs 3 days after transfection with Luc or Pkm2 modRNA with scrambled siRNA or Pkm2 modRNA with siRNA for β -catenin (n=3). One-way ANOVA, Tukey's Multiple Comparison Test for A&B, H&I, L&M. Unpaired two-tailed t-test for E. ***, P<0.001, **, P<0.01, *, P<0.05, N.S, Not Significant. Scale bar = 10μm (D) and 25μm (G&K).