Research Paper Volume 11, Issue 24 pp 12546—12567

Sirt1-inducible deacetylation of p21 promotes cardiomyocyte proliferation

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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.