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. 2014 Nov 20;3(6):1103–1117. doi: 10.1016/j.stemcr.2014.10.006

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© 2014 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

PMC Copyright notice

miR-590 Regulates the SSB Damage Repair, DSB Damage Repair, and Proliferation of mESCs by Acvr2a

(A) Morphology of mESCs clones overexpressing Acvr2a. The scale bar represents 100 μm.

(B) Analysis of proliferation of mESCs overexpressed with Acvr2a by MTS cell proliferation assay. Data shown are means ± SD of five independent experiments (n = 5). ^∗∗∗p < 0.001.

(C) Analysis of proliferation of mESCs overexpressed with Acvr2a by FACS analysis of BrdU incorporation. The figure means the percentage of the population of BrdU-positive cells. Data shown are means ± SD of three independent experiments (n = 3). ^∗∗∗p < 0.001.

(D) Comet assay for SSB damage in mESCs. The scale bar represents 100 μm. Right panel is the quantification of average DNA tail moment. Data shown are means ± SD of three independent experiments (n = 3). ^∗∗∗p < 0.001.

(E) Immunofluorescence analysis of γ-H2AX (red) to indicate the state of DSB damage in mESCs. Ho.33342 (Hoechst 33342) represents nuclear staining (blue). The scale bar represents 100 μm. (Bottom) The quantification of the fluorescence of γ-H2AX immunostaining. Data shown are means ± SD of three independent experiments (n = 3). ^∗∗∗p < 0.001.

(F) qRT-PCR verification of transcript levels of the cell cycle regulation and homologous recombination damage repair genes in mESCs. The right side shows the protein levels according to western blot analysis. Data shown are means ± SD of three independent experiments (n = 3). ^∗p < 0.05 and ^∗∗p < 0.01.

(G) Morphology of mESCs clones. The scale bar represents 100 μm.

(H) Cell proliferation assay in the rescue experiment by MTS cell proliferation assay. Data shown are means ± SD of three independent experiments (n = 3). ^∗∗∗p < 0.001.

(I) Cell proliferation assay in the rescue experiment by FACS of BrdU incorporation. The figure means the percentage of the population of BrdU-positive cells. Data shown are means ± SD of three independent experiments (n = 3). ^∗∗p < 0.01.

(J) Comet assay shows the SSB damage state. The scale bar represents 100 μm. The right panel is the quantification of average DNA tail moment. Data shown are means ± SD of three independent experiments (n = 3). ^∗∗∗p < 0.001.

(K) Overexpression of ACVR2a restores DSB damage in mESCs transfected with pre-miR-590. Immunofluorescence analysis of γ-H2AX (red) indicates the state of DSB damage in mESCs with Ho. 33342 (Hoechst 33342) staining for the nucleus (blue). The scale bar represents 100 μm. (Bottom) The quantification of the fluorescence of γ-H2AX immunostaining. Data shown are means ± SD of three independent experiments (n = 3). ^∗∗p < 0.01.

(L) qRT-PCR and western blot analysis of the cell cycle- and homologous recombination damage repair-related genes in mESCs. Data shown are means ± SD of three independent experiments (n = 3). ^∗p < 0.05 and ^∗∗p < 0.01.