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. 2019 Nov 28;9:17865. doi: 10.1038/s41598-019-54334-4

Figure 2.

Figure 2

Hypoxia forces CMs to enter an incomplete cell cycle and thus increases ploidy and nuclear numbers in vivo. (a) Representative distribution of ploidy of cardiomyocytes in both normoxic and hypoxic group (scale bars, 100 μm). The white arrow head indicates trinucleated and tetranucleated cardiomyocytes. (b) The percent of polyploid cardiomyocytes between normoxic and hypoxic group (n = 3 each). (c) The mean ploidy of cardiomyocytes (n = 3 each). (d) The percent of multinucleated cardiomyocytes between acyanotic and cyanotic group (n = 6 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal). (e) The mean nuclear number of cardiomyocytes in both acyanotic and cyanotic group (n = 6 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal). (f) WGA staining in both acyanotic and cyanotic group. Results are displayed as two representative images in both group (upper, scale bars, 50 μm), as two bar charts of relative area of cardiomyocytes (lower left, n = 3 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal) and relative length of cardiomyocytes (lower right, n = 3 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal). (g) Co-immunostaining with WGA staining, anti-Ki67 and anti-cTnT antibodies. Results are displayed as a presentative image (left, scale bars, 25 μm), as two bar charts of Ki67-positive cardiomyocyte nuclei (upper right, n = 6 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal) and Ki67-positive cardiomyocytes (lower right, n = 3 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal). (h) Co-immunostaining with anti-pH3 and anti-cTnT antibodies. Results are displayed as a presentative image (left, scale bars, 25 μm) and as a bar chart (right, n = 4 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal). (i) Co-immunostaining with anti-Aurora B and anti-cTnT antibodies. Results are displayed as a representative image (left, scale bars, 25 μm) and as a bar chart (right, n = 3 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal). (j) Co-immunostaining with anti-mklp2 and anti-cTnT antibodies. Results are displayed as a representative image (left, scale bars, 25 μm) and as a chart (right, n = 4 each, >600 cardiomyocytes from 10 randomly chosen fields were counted for each individual animal). Data is presented as mean ± s.d. *P < 0.05.