Figure 5
From: Fam64a is a novel cell cycle promoter of hypoxic fetal cardiomyocytes in mice
Fam64a expression positively correlates with fetal cardiomyocyte (fCM) proliferation. (a) qPCR analysis of mRNA levels of the indicated genes in Fam64a-silenced fCMs (E16–E17) isolated under low O2 conditions. n = 3 independent experiments except for Fam64a knockdown evaluated with 2 independent experiments. ** P < 0.01 and *** P < 0.001 compared to the si-control levels of each gene. (b) Immunofluorescence staining for Ki67 observed in α-actinin and DAPI of Fam64a-silenced E16 fCMs isolated under low O2 conditions. Arrows denote Ki67-positive CMs. Quantitative analysis of the proportion of Ki67-positive CMs is shown on the right. n = 5 independent experiments. * P < 0.05. (c) Proliferative activity of Fam64a-silenced fCMs (E16–E17) evaluated by cell counting. The cells were isolated under low O2 conditions and cultured for 48–96 h. n = 5 independent experiments. * P < 0.05. (d) Percentage of Fam64a-silenced E16 fCMs that completed cell division, as determined by time-lapse imaging. The cells were isolated under low O2 conditions. n = 3 independent experiments and ~700 cells were counted for each condition. * P < 0.05. (e) qPCR analysis of Fam64a mRNA expression in fCMs (E15–E16) isolated under high O2 conditions and transduced with a Fam64a-GFP-baculovirus or a control GFP-expressing baculovirus. n = 3 independent experiments. (f) Immunoblot analysis of fCMs (E15–E16) isolated under high O2 conditions and transduced with a Fam64a-GFP-baculovirus or a control GFP-expressing baculovirus (denoted as F-G and G, respectively). A GFP antibody was used as this correctly detected overexpressed Fam64a (28 kDa) as a GFP-tagged protein (28 + 27 kDa). The band specificity was confirmed by co-transfection of siRNA targeting GFP at the indicated doses. (g) Localization of transduced Fam64a-GFP protein in fCMs and fibroblasts, as observed in bright-field images. (h) Percentage of Fam64a-overexpressed fCMs (E14–E17) that completed cell division, as determined by time-lapse imaging. The cells were isolated under high O2 conditions. n = 4–5 independent experiments and ~1800 cells were counted for each condition. * P < 0.05. Error bars = SEM. Scale bars = 50 µm in (b) and 30 µm in (g).
