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. 2022 Dec 20;13:7672. doi: 10.1038/s41467-022-35286-2

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© The Author(s) 2022

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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Fig. 3 — a Nanog STREAMING-tag knock-in (NSt) cells transiently expressing TetR(WT)-mNG and MCP-RFP. In cells with high TetR(WT)-mNG expression (right), MCP spots that overlapped with TetR(WT) spots were rarely observed (closed arrowhead). In cells with low TetR(WT)-mNG expression (left), MCP spots tended to overlap with TetR(WT) spots (open arrowhead). Dashed line indicates the cell nucleus. Scale bar, 5 µm. b Percentage of cells in which TetR and MCP spots were simultaneously visible and colocalized. Mean values with SD of three biological replicates (>30 cells) are shown. P-values correspond to unpaired, two-sided Student’s t-test. c 3D structure of TetR (PDB 1QPI) and mutation site location. d Images of NSt cells co-transfected with TetR(W43F)-mNG and MCP-RFP. Open arrowheads indicate TetR(W43F) spots with MCP spots. Dashed line indicates the cell nucleus. Scale bar, 5 µm. e Distribution of Nanog mRNA counts in NSt derived cell lines expressing MCP-RFP and either NLS-mNG (NLS), TetR(W43F)-mNG (TetR(W43F)), or TetR(WT)-mNG (TetR(WT)). Box plots indicate the interquartile range IQR (25–75%) with a line at the median. Whiskers indicate 1.5 times the IQR. n, number of cells analyzed. P-values were determined using two-sided Wilcoxon rank sum test. f Distribution of relative fluorescence intensity at MCP transcription sites in cells expressing NLS, TetR(W43F) and TetR(WT). n, number of cells analyzed. P-values were determined using two-sided Wilcoxon rank sum test. g Bar graph showing the percentage of cells with STREAMING-tag knock-in allele in the ON state in NLS, TetR(W43F) and TetR(WT)-expressing cells. Data are presented as the means of three biological replicates (more than 33 cells per experiment). The error bars indicate SD. P-values were determined using unpaired, two-sided Student’s t-test. h RNAPII ChIP-qPCR analysis of three locations on STREAMING-tag in NLS, TetR(W43F) and TetR(WT)-expressing cells. Data are presented as the means of n = 2 biological replicates.