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. 2020 Jan 30;48(6):2924–2941. doi: 10.1093/nar/gkaa051

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© The Author(s) 2020. 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 Non-Commercial 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 7. — Depletion of WDR5 phenocopies WIN site inhibition. (A) Immunoblotting for WDR5 in CHP-134 cells in which endogenous WDR5 is tagged with an FKBP12^(F36V)–HA cassette. The blot for WDR5 shows that its molecular weight is shifted upon introduction of the cassette in tagged cell populations, compared to untagged CHP-134 cells (middle panel). Addition of 500 nM of dTAG-47 ligand induces disappearance of tagged WDR5, as judged by both the total WDR5 blot and the anti-HA blot (top panel), which visualizes only tagged WDR5. GAPDH is a loading control. (B) Distribution of cell cycle phases as determined by flow cytometry of tagged CHP-134 cells treated with DMSO or 500 nM dTAG-47 (dT47) for 2, 4 or 7 days. Data are presented as mean, error bars are standard error. *P = 0.01, **P = 0.005, ***P = 0.0003, ****P < 0.0001 by Student's t-test. n = 3. (C) Immunoblotting of steady-state levels of the indicated proteins in tagged CHP-134 cells that were treated with DMSO or 500 nM dTAG-47 for 2 days. GAPDH is a loading control. (D) Table shows the number of transcripts significantly (FDR < 0.05) altered (in RNA-Seq analysis) by one day of treatment of tagged CHP-134 cells with 500 nM dTAG-47, compared to DMSO control. (E) GO enrichment clusters for gene transcripts significantly repressed by dTAG-47 in modified CHP-134 cells, as determined by RNA-Seq. Numbers in italics represent the number of repressed genes in each category. Categories in yellow are Biological Process; categories in orange are Molecular Function. (F) Venn diagrams, showing overlap of genes repressed (top) or induced (bottom) by degradation of WDR5 in CHP-134 cells with the 94 genes bound by WDR5 across all six cell types. (G) Venn diagrams, showing the overlap of gene expression changes (FDR < 0.05) induced by degradation of WDR5 (dTAG) or chemical blockade of the WIN site (C6) in CHP-134 cells. Diagrams are broken down into repressed or induced genes. (H) GSEA, comparing genes repressed by dTAG-mediated degradation of WDR5 in CHP-134 cells versus C6-treatment of CHP-134 cells. (I) Top GO enrichment categories for genes repressed by both C6 and dTAG-47 (common repressed), induced by both C6 and dTAG-47 (common induced), repressed by C6 but not dTAG-47 (C6 repressed), induced by C6 but not dTAG-47 (C6 induced), repressed by dTAG-47 but bot C6 (dT47 repressed), and induced by dTAG-47 but bot C6 (dT47 induced). Numbers to the right of the graph indicate the number of genes in each category.