Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 Dec 18;9:5375. doi: 10.1038/s41467-018-07787-6

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/.

PMC Copyright notice

Fig. 7 — Rybp and Eto2 knock-down phenocopies Scl^-/- cardiac phenotype. a Outline of functional assays. b–e siRNA-mediated Eto2 (siEto2) and Rybp (siRybp) knock-down in day 3.5 EBs. b qRT-PCR analysis of Eto2 and Rybp mRNA levels. Analysis from day 3.5 WT and Scl^-/- EB cells is shown for comparison. siNeg, siRNA negative control; n = 4. c Western blot analysis of RYBP and H2AK119ub levels. mSIN3A and H2A, loading controls; n = 2. d, e Day 3.5 WT, Scl^-/-, siEto2-treated, siRybp-treated and siNeg-treated EB cells were plated in cardiac condition. cTNT expression was monitored at day 7 by IF (d, top) and intra-cellular FACS (d, bottom). Quantitation of FACS data from Scl^-/-, siEto2- and siRybp-treated cultures is shown as Scl^-/-/WT, siEto2/siNeg and siRybp/siNeg ratios (e); n = 2. Scale bars, 100 μm. f qRT-PCR analysis of Rybp expression in day 3.5 untreated (UN), tamoxifen- (TAM) and ethanol (EtOH)-treated CreERT2:Rybp^fl/fl EBs; n = 2. g Day 3.5 untreated, EtOH-treated or TAM-treated CreERT2:Rybp^fl/fl cells were plated in cardiac condition. cTNT expression was assessed at day 7 by IF (green, left) and qRT-PCR analysis (Tnnt2, shown as TAM/ETOH fold increase, right). Scale bar, 100 μm. n = 2. h Day 3.5 WT, Scl^-/-, siEto2-treated, siRybp-treated and siNeg-treated EB cells were plated in blast colony assay; n = 2. i Western blot analysis of H2AK119ub in day 3.5 EB cells treated with increasing concentrations of PRC1 inhibitor (PRT4165). UNT, untreated; αTubulin and H2A, loading controls, n = 2. j PRC1 inhibitor or DMSO-treated EBs were plated in cardiac assay, and cTNT expression assessed at day 7 by IF (green, top), FACS (bottom) and qRT-PCR (Tnnt2, right); n = 3. k Western blot analysis of H3K27me3 in day 3.5 EB cells treated with increasing concentrations of PRC2 inhibitor or analogue. mSIN3A, loading control, n = 2. l Day 3.5 EB cells treated with PRC2 inhibitor or analogue were plated in cardiac condition, and cTNT expression assessed at day 7 by IF (green, left) and qRT-PCR (Tnnt2, right). n = 2. Scale bars, 100 μm. Mean ± SD is shown in b, f, h, j, l; mean of ratios of mutant samples versus controls ± SD is shown in e, g; student’s t-test, *p < 0.05. See also Supplementary Fig. 8