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. 2016 Apr 21;44(14):6741–6755. doi: 10.1093/nar/gkw301

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© The Author 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — JMJD3 regulates cardiac differentiation. (A) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). (B) PCR genotyping of WT and Jmjd3^+/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3-KO ESCs. (C) Relative mRNA expression of Jmjd3 in wild type and Jmjd3^+/− ESCs using primers specific target to exon 9–10. (D) Relative mRNA expression of pluripotency genes (Oct4, Sox2 and Nanog) in wild type and Jmjd3^+/− ESCs. (E) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes (Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20) in day 7 EBs differentiated from wild type and Jmjd3^+/− ESCs. (F) Relative mRNA expression of cardiomyocyte genes (α-MHC and β-MHC) in day 12 EBs differentiated from wild type and Jmjd3^+/− ESCs. (G) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3^+/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20. ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. (H) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes (Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20) in day 7 EBs differentiated from ishJmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. (I) Relative mRNA expression of cardiomyocyte genes (α-MHC and β-MHC) in day 12 EBs differentiated from ishJmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. (J) Percentage of beating EBs in day 12 EBs differentiated from ishJmjd3 in the presence or absence of Dox. (K) Western blot analysis of total protein extracts of day 12 EBs differentiated from ishJmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.