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. 2018 Aug 17;8:12337. doi: 10.1038/s41598-018-30886-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Nuclear novex-3 is a cell cycle promoter in hypoxic fCMs. (a) Novex-3 was silenced in E17 fCMs by siRNA, and the nuclear fraction was used to probe novex-3 protein by immunoblotting. Two siRNAs specific for novex-3, designated as si-1 and si-2, were used. Histone H3 was included as a loading control. (b) qPCR analysis of mRNA levels of the indicated genes in novex-3-silenced fCMs (E16–E17). Data are shown as normalised to the si-control level of each gene, set at 1. n = 3 independent experiments. ***P < 0.001 compared to the si-control level of each gene. (c,d) Immunofluorescence for Ki67 (c, green) and phospho-histone H3 (pH3) (d), green) observed in sarcomeric α-actinin (red, as a CM marker) and DAPI (blue) in novex-3-silenced fCMs (E17). Arrows denote Ki67-positive (c) and pH3-positive (d) fCMs. Proportions of Ki67-positive (c) and pH3-positive (d) fCMs were shown on the right. n = 4 independent experiments. *P < 0.05 compared to si-control. Scale bars = 50 µm. (e) Proliferative activity of novex-3-silenced E16–E17 fCMs evaluated by cell counting after 72–96 h culture. n = 3 independent experiments. **P < 0.01 compared to si-control. (f,g) Immunofluorescence for Ki67 (f), green) and phospho-histone H3 (pH3) (g, green) observed in sarcomeric α-actinin (red, as a CM marker) and DAPI (blue) in novex-3-1-overexpressing fCMs (E15–E17). Arrows denote Ki67-positive (f) and pH3-positive (g) fCMs. Proportions of Ki67-positive (f) and pH3-positive (g) fCMs are shown at right. n = 4 independent experiments. *P < 0.05 compared to control GFP-overexpressing fCMs. Scale bars = 50 µm. (h) Percentage of novex-3-1-overexpressing fCMs that completed cell division, as determined by time-lapse imaging. Analysis was performed separately in two developmental stages (E15–E17 and later than E18). n = 3 independent experiments. *P < 0.05 compared to control GFP-overexpressing fCMs. (i) Left, overexpression of novex-3-1-GFP mRNA in E17 fCMs was confirmed by probing 61 bp amplicon within GFP sequence by PCR. Right, overexpression of novex-3-1-GFP protein in E17 fCMs was confirmed by immunoblotting the whole protein extracts using the antibody against GFP. A clear single band was observed at the expected molecular mass (novex-3-1: ~175 kDa + GFP: ~27 kDa). β-tubulin was included as a loading control. Error bars = SEM.