Research Paper Volume 11, Issue 24 pp 12546—12567

Figure 4. Sirt1 induces CM proliferation in vitro. (A) Western blotting and quantitative analyses of Sirt1 levels in E16.5, P1 and P28 mouse hearts. β-actin was used as a loading control (n=5). (B) Immunofluorescence of pH3 in P1 CMs transfected with Ad-NC or Ad-Sirt1 and quantification of pH3-positive CMs. pH3-positive CMs are indicated by arrows. Scale bar, left, 100 μm, right, 20 μm. Quantitative analyses are representative of fields from 5 mice per group. (C) Immunofluorescence of Aurora B in P1 CMs transfected with Ad-NC or Ad-Sirt1 and Aurora B-positive CMs were quantified. The arrow indicates a CM cell division, in which Aurora B localizes at the midbody during cytokinesis. Scale bar, 20 μm. Quantitative analyses are representative of fields from 5 mice per group. (D) Immunofluorescence was performed for cTnT and quantification of the counts of P1 CMs transfected with Ad-NC or Ad-Sirt1 (n=5), Scale bar, 500 μm. (E) Western blotting and quantitative analyses of Sirt1 levels in P6 CMs transfected with Ad-NC or Ad-Sirt1. β-actin was used as a loading control (n=5). (F) Immunofluorescence for EdU in P6 CMs transfected with Ad-NC or Ad-Sirt1. EdU-positive CMs were quantified. Scale bar, 50 μm. EdU-positive CMs are indicated with arrows. Quantitative analyses are representative of fields from 5 mice per group. (G) Immunofluorescence for Aurora B in P6 CMs transfected with Ad-NC or Ad-Sirt1. Aurora B-positive CMs were quantified. The arrow indicates a CM cell division, in which Aurora B localizes at the midbody in cytokinesis. Scale bar, 50 μm. Quantitative analyses are representative of fields from 5 mice per group. (H) Immunofluorescence for cTnT and quantification of the counts of P6 CMs transfected with Ad-NC or Ad-Sirt1 (n=5), Scale bar, 500 μm. (I) DNA synthesis was assessed using EdU immunofluorescence staining and EdU-positive CMs were quantified in Sirt1-silenced P1 CMs. Scale bar, up, 100 μm, down, 20μm. Quantitative analyses are representative of fields from 5 mice per group. (J) Mitosis was detected using pH3 immunofluorescence staining and pH3-positive CMs were quantified in Sirt1-silenced P1 CMs. pH3-positive CMs are indicated by arrows. Scale bar, up, 100 μm, down, 20μm. Quantitative analyses are representative of multiple fields from 5 mice per group. (K) Immunofluorescence for cTnT and quantification of the counts of P1 CMs transfected with si-NC or si-Sirt1 (n=5), Scale bar, 500 μm. (L) Western blotting and quantitative analyses of Sirt1 levels in P6 CMs transfected with si-NC or si-Sirt1. β-actin was used as a loading control (n=5). (M) Immunofluorescence for EdU and quantification of EdU-positive CMs in P6 CMs transfected with si-NC or si-Sirt1. The arrow indicates EdU-positive CM nuclei. Scale bar, 50 μm. Quantitative analyses are representative of fields from 5 mice per group. (N) Immunofluorescence for cTnT and quantification of the counts of P6 CMs transfected with si-NC or si-Sirt1 (n=5), Scale bar, 500 μm. Statistical significance was calculated using a one-way ANOVA followed by the LSD post hoc test in A and two-tailed unpaired Student’s t-test in B-N. *p<0.05; data are presented as the mean ± S.E.M.