23064 nanog  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Pioneer activity distinguishes activating from non-activating pluripotency transcription factor binding sites

doi: 10.1101/2022.07.27.501606

Figure Lengend Snippet: (A) Schematic representation of the dTAG system, wherein an FKBP-tagged protein can be proteasomally degraded upon addition of the dTAG-13 small molecule. (B) Western blot showing protein abundance of SOX2 and NANOG upon addition of dTAG-13 for the indicated times in respective FKBP-tagged cell lines. HSP90 was used as loading control. NT = Not Treated. (C) Quantitative mass spectrometry results showing the differential protein abundance upon 30 minutes of dTAG treatment versus DMSO treated SOX2-FKBP cells and parental (untagged) cells. (D) Brightfield images showing colony morphology upon addition of dTAG-13 for the indicated timepoints. (E) Genomic tracks showing accessibility by ATACseq around the Ctgf gene for FKBP-tagged cell lines after indicated times of depletion by dTAG treatment. Y-axes show reads per genomic content (RPGC) (F) The number of differentially accessible peaks shown for every FKBP-tagged cell line and time of depletion. Positive bars indicate peaks with increased accessibility (gain), negative bars peaks with decreased accessibility (loss). Colour intensity is indicative of effect size of change in accessibility.

Article Snippet: The blots were incubated with the following primary antibodies overnight at 4 °C: SOX2 (1:1000, D9B8N, Cell Signaling), OCT4 (1:1000, D6C8T, Cell Signaling), NANOG (1:1000, D2A3, Cell Signaling), GFP (1:1000, ab6673, abcam), and as loading control HSP90 (1:2000, 13171-1-AP).

Techniques: Western Blot, Quantitative Proteomics, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: Pioneer activity distinguishes activating from non-activating pluripotency transcription factor binding sites

doi: 10.1101/2022.07.27.501606

Figure Lengend Snippet: (A) Quantitative mass spectrometry results showing the differential protein abundance comparing 30 minutes of dTAG-13 treated and untreated NANOG-FKBP and OCT4-FKBP cells. (B) Principal component analysis (PCA) scatter plot of accessibility data of the three FKBP-tagged lines indicating the first two principal components (PCs) (left). Arrows indicate major movements over time. On the right, the amount of variance explained by each principal component. (C) Tornado plot showing accessibility in the SOX2-, OCT4- and NANOG-FKBP-lines in untreated cells and following 2 hours of dTAG-13 treatment at differentially accessible regions for the indicated lines. Note that the ‘Up’ category in the NANOG-FKBP has been omitted for having too few peaks.

Article Snippet: The blots were incubated with the following primary antibodies overnight at 4 °C: SOX2 (1:1000, D9B8N, Cell Signaling), OCT4 (1:1000, D6C8T, Cell Signaling), NANOG (1:1000, D2A3, Cell Signaling), GFP (1:1000, ab6673, abcam), and as loading control HSP90 (1:2000, 13171-1-AP).

Techniques: Mass Spectrometry, Quantitative Proteomics

Journal: bioRxiv

Article Title: Pioneer activity distinguishes activating from non-activating pluripotency transcription factor binding sites

doi: 10.1101/2022.07.27.501606

Figure Lengend Snippet: (A) Genomic tracks showing accessibility by ATACseq in untreated cells and after 2 hours of depletion in for SOX2-, OCT4- and NANOG-FKBP-tagged cell lines. Examples of sites where accessibility is regulated by both, or exclusively, SOX2 or OCT4 are shown. Y-axes show reads per genomic content (RPGC) (B) Scatter plot showing the effect sizes at peak locations following 2 hours of SOX2 depletion against 2 hours of OCT4 depletion. Colours indicate categorisation of differentially accessible regions (DARs) as mentioned in the main text. (C) Heatmap showing the enrichment and depletion of TF binding motifs in the DARs. Shown are the top 25 motifs with the lowest enrichment p-value in any category. Log odds ratio was calculated as coefficients in logistic regression with DAR category membership as dependent variable, controlling for the GC-bias and total motif presence in peaks. Significance was tested using a single-parameter Wald-test in the same regression. (D) Tornado plots showing accessibility at peaks belonging to the DAR categories in the SOX2 and OCT4 degron cell lines. (E) Quantification of the heights of peaks belonging to the DAR categories over time of depletion in the SOX2 and OCT4 degron lines. Shaded area indicates the first quartile (Q1) up to the third quartile (Q3) of heights, whereas points indicate the median (Q2).

Article Snippet: The blots were incubated with the following primary antibodies overnight at 4 °C: SOX2 (1:1000, D9B8N, Cell Signaling), OCT4 (1:1000, D6C8T, Cell Signaling), NANOG (1:1000, D2A3, Cell Signaling), GFP (1:1000, ab6673, abcam), and as loading control HSP90 (1:2000, 13171-1-AP).

Techniques: Binding Assay