Skip to main content
. 2016 Feb 7;110(1):73–84. doi: 10.1093/cvr/cvw031

Figure 4.

Figure 4

miR-26a controls proliferation in neonatal cardiomyocytes. (A) Expression of miR-26a in neonatal and adult cardiac myocytes (black bars) and non-myocyte cells (white bars); data are expressed as mean ± SEM; **P < 0.01, n = 5. (B) Time course analysis of miR-26a expression in neonatal cardiomyocytes transfected with scrambled LNA (LNA scr; white bars) or anti-miR-26a LNA inhibitor (LNA miR26a; black bars). (C) Expression of Ccne1, Cdk6, and Ezh2 in neonatal cardiomyocytes transfected with LNA scr (white bars) or LNA miR26a (black bars). (D) Western blot analysis of Ezh2 and Ccne1 expression in neonatal cardiomyocytes transfected with LNA scr or LNA miR26a. (E) Expression of Cdkn1a, Cdkn2a, and Cdkn2b in neonatal cardiomyocytes transfected with LNA scr (white bars) or LNA miR26a (black bars). Data are expressed as mean ± SEM; n ≥ 3; *P < 0.05; **P < 0.01. (F) In upper panels, immunostaining of BrdU-positive cells (red) transfected with LNA miR26a or with scrambled LNA; cardiomyocytes are identified by α-actinin staining (green) and nuclei with DAPI (blue). In the lower panels, the bar graphs represent the percentage of total BrdU-positive cells (left), the percentage of cardiomyocytes (middle) and the percentage non-myocytes (right) that are BrdU-positive. Data are mean ± SEM of 56 fields per group; *P < 0.05, **P < 0.01. Scale bar: 50 μm. (G) In upper panels, immunostaining of phosphohistone 3 (PH3)-positive cells (red) transfected with LNA miR26a or scrambled LNA scr (upper panel) or LNA miR26a (lower panel); cardiomyocytes are identified by α-actinin staining (green) and nuclei with Hoechst (blue). In lower panels, bar graphs show the percentage of PH3-positive total cells (left), the percentage of cardiomyocytes (middle) and of non-myocytes (right). Data are expressed as mean ± SEM of per cent PH3-positive cardiomyocytes in a minimum of 25 micrographs taken at ×40 magnification per group; *P < 0.05, **P < 0.01. Scale bar: 50 μm. (H) Cardiomyocyte cultures were transfected with either LNA miR26a or with scrambled LNA and stained with α-actinin antibody. The bar graph shows average number ± SEM of cardiomyocytes per ×20 field in triplicate cultures (56 fields each); **P < 0.01. (I) Analysis of the cell cycle in neonatal cardiomyocytes following propidium iodide staining and cytometry 6 days after transfection with LNA scr (white bars) or LNA miR26a (black bars). An example of S-phase analysis is depicted in the left panel. Data are expressed as mean ± SEM; n ≥ 3; *P < 0.05; **P < 0.01. See also Supplementary material online, Figure S5.