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. 2018 Jan 19;8:1189. doi: 10.1038/s41598-017-19114-y

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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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POU5F1 knockdown facilitates synMYOD1-induced myogenic gene activation. (a) Immunostaining analysis for POU5F1 in the siPOU5F1 and synMYOD1 (siPOU5F1/synMYOD1)-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. (b) Immunostaining analysis for NANOG in the siPOU5F1/synMYOD1-transfected cells at day 0 to day 3 post transfection. MYOD1 was detected by the specific antibody. Nuclei were stained with DAPI. Scale bar: 10 μm. (c) Immunoblotting analysis for POU5F1 in the siPOU5F1/synMYOD1-transfected cells at day 3 post transfection. MYOD1 was detected by the specific antibody. The H3 antibody was used as a loading control. The relative intensities of POU5F1 signals normalized by H3 were compared between no transfection and siPOU5F1/synMYOD1 transfection (mean ± SEM from three independent biological replicates). *P < 0.01, t-test. (d) ChIP analysis showing POU5F1 enrichment at the promoter regions (Pro-1 and Pro-2) of POU5F1 in hESCs, siControl/synMYOD1-treated cells, and siPOU5F1/synMYOD1-treated cells. The promoter regions of MEF2C and MYOG were used as negative controls. The error bars indicate the SEM from three independent biological replicates. *P < 0.05, t-test. (e) ChIP analysis showing MYOD1 enrichment at the promoter regions of MEF2C and MYOG in hESCs, siControl/synMYOD1-treated cells, and siPOU5F1/synMYOD1-treated cells. mRNA encoding HA-tagged MYOD1 and anti-HA antibody was used in this experiment as the anti-MYOD1 antibody suitable for ChIP was not available. The promoter regions of POU5F1 were used as negative controls. The error bars indicate the SEM from two independent biological replicates. *P < 0.05, t-test. NS: not significant. (f) qPT-PCR analysis for the expression of myogenic markers in the siControl (siC), siPOU5F1 (siP), siC/synMYOD1, and siP/synMYOD1-treated cells. The expression levels were normalized to GAPDH. The error bars indicate the SEM from two independent biological replicates. *P < 0.01, t-test. Uncropped images of the blots for Fig. 2c are shown in Supplementary Figure 7.