ctcf rabbit  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: CTCF maintains pericentromere function and mitotic fidelity

doi: 10.1101/2025.05.30.657091

Figure Lengend Snippet: (A) Metaphase example images of CENP-E immunofluorescence (red) and DNA stained with Hoechst (cyan) in untreated (Unt), CTCF KD via 3-day 5-Ph-IAA treatment, and CENP-E inhibitor GSK-923295 treatment 10nm for 1 day. Scale bar is 10 µm. (B) Graph of CENP-E relative intensity on metaphase plate chromosomes in untreated, CTCF KD, and GSK- 923295. Data are from 3 biological replicates with n>12 for each replicate. (C) Graph of the percentage of polar chromosomes and categorizations of single (green) or multiple (orange) polar chromosome pairs present in untreated, CTCF KD, and GSK-923295. Data for polar chromosomes represents three replicates with n>15 for each replicate. Polar chromosomes are present in untreated (3/69), 3-day CTCF KD (5/27), GSK-923295 (63/98). Statistical tests for panel B and C ANOVAs followed by Post Hoc Tukey tests. Error bars represent standard error. Significance is represented by *p < 0.05, **p < 0.01, and ***p < 0.001, and ns represents no statistical significance.

Article Snippet: The primary antibodies used were CTCF Rabbit (2899, Cell Signaling Technologies) 1:100, CENP-E mouse (39619, Active Motif) 1:1000, ACA human (HCT-0100 Immunovision) 1:4000, CST) 1:1000.

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Genome reorganization and its functional impact during breast cancer progression

doi: 10.1101/2025.05.14.654144

Figure Lengend Snippet: Reorganization of compartments, TADs, and loops during breast cancer progression (A) A diagram of the experimental design. Three epithelial cell lines represent various stages of breast cancer progression; MCF10A are non-cancerous, MCF10AT1 are pre-malignant, and MCF10CA1a are metastatic. In each cell line we generated 5kb resolution Micro-C to identify features such as compartments, topologically associating domains (TADs), and chromatin loops. We overlapped these features with functional changes in gene expression from RNA-Seq, histone modifications and CTCF binding from ChIP-Seq, and chromatin accessibility from ATAC-Seq. (B) Micro-C maps of a 2 Mb region of chromosome 1 in MCF10A (non-cancerous), MCF10AT1 (pre-cancerous), and MCF10CA1a (metastatic) cells at 5 kb resolution. Each map has annotations for loop calls, both static (black boxes) and diYerential (red boxes). Below each map is a track indicating compartment calls from CALDER (dark red is most A-like, dark blue is most B-like) as well as insulation scores tracks with static (grey) and diYerential (red) boundaries marked. Ribbons indicate TAD calls for each cell type. (C) Lengths of the genome assigned to each compartment in each cell type. (D) TAD and (E) loop calls from each cell type, colored by the number of maps they were initially detected in. (F) Saddle plots of interactions between regions of diYerent compartments in MCF10A, MCF10AT1, and MCF10CA1a. Bottom plots indicate the average eigenvector value for each compartment ventile. Plots shown are for chromosomes 2, 12, and 17 (see Methods). (G) Left; DiYerential TAD boundaries clustered by their timing of change, depicted in line plots and heatmap. Right; aggregate plots of weakened and strengthened TAD boundaries (n=100). (H) Left; DiYerential chromatin loops clustered by their timing of change, depicted in line plots and heatmap. Right; aggregate plots of weakened and strengthened loops (n=100).

Article Snippet: ChIPseq for CTCF (Cell Signaling Technology, catalog number 3418) and histone marks H3K27ac (Abcam, ab4729) and H3K27Me3 (Abcam ab6002).

Techniques: Generated, Functional Assay, Gene Expression, RNA Sequencing, Binding Assay, ChIP-sequencing, Insulation

Journal: bioRxiv

Article Title: Genome reorganization and its functional impact during breast cancer progression

doi: 10.1101/2025.05.14.654144

Figure Lengend Snippet: Persistent chromatin loops connect diVerentially expressed genes to distal enhancers. (A) Percentages of loops designated as either promoter-promoter, enhancer-promoter, enhancer-enhancer, or single-sided promoter or enhancer loops. (B) Distributions of loop sizes by enhancer/promoter designations. P-values represent T-tests comparing the means of diYerent loop classes. Boxplots show the median (middle line), 25 th and 75 th quartiles (box perimeters), and range excluding outliers (dashed line whiskers). Outliers are defined as values that are over 1.5 times the interquartile range beyond the box bounds and are excluded from these plots. (C) Distributions of loop strength by enhancer/promoter designations. (D) Percentages of upregulated genes that have gained H3K27ac at promoters, distal enhancers, both, or gained loops. P-values represent T-tests comparing the means of various loop sets. Non-significant (n.s.) represents p-values above 0.05. (E) Log2 fold-change of distal H3K27me3 (grey), distal H3K27ac (red), promoter H3K27ac (orange), gene expression (yellow), and loop strength (blue), when overlapped. Grey dots indicate features that do not change significantly, while colored points are significantly diYerential features. Boxplots are defined as in (B). P-values represent T-tests comparing the means of each class to 0. Non-significant (n.s.) represents p-values above 0.01. (F) Percentages of downregulated genes that have gained H3K27ac at promoters, distal enhancers, both, or gained loops. (G) Log2 fold-change of distal H3K27me3 (grey), distal H3K27ac (red), promoter H3K27ac (orange), gene expression (yellow), and loop strength (blue), when overlapped. Boxplot details as defined in (E). (H) An example of an upregulated gene (SPRY1) connected to gained enhancers by static loops. Black boxes show loop annotations. Red compartment tracks indicate A compartments, while blue indicates B compartments. In CTCF signal tracks, red highlights indicate diYerential CTCF peaks. In H3K27ac and ATAC-Seq signal tracks, red highlights indicate diYerential enhancers as determined by changes in H3K27ac. Genes highlighted in black are diYerentially expressed. (I) An example of downregulated genes (SCNN1G, SCNN1B) connected to lost enhancers by static loops. Plot annotations are as described in (H).

Article Snippet: ChIPseq for CTCF (Cell Signaling Technology, catalog number 3418) and histone marks H3K27ac (Abcam, ab4729) and H3K27Me3 (Abcam ab6002).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Genome reorganization and its functional impact during breast cancer progression

doi: 10.1101/2025.05.14.654144

Figure Lengend Snippet: DiVerential loops are enriched for cancer-relevant diVerentially expressed genes (A) Log2 fold-change of diYerentially expressed genes at the anchors of gained (blue), weakened (green), or static (grey) loops. Boxplots show the median (middle line), 25 th and 75 th quartiles (box perimeters), and range excluding outliers (dashed line whiskers). Outliers are defined as values that are over 1.5 times the interquartile range beyond the box bounds and are excluded from these plots. P-values represent T-tests comparing the mean of each set to 0. (B) Bar plot showing the number of diYerentially expressed genes at strengthened or weakened loop anchors. Bar segments are colored by whether the gene is changing the same (blue for upregulated in strengthened loops, green for downregulated in weakened loops) or opposite (grey) direction as the loop. P-value represents a Fisher’s Exact Test for whether the odds ratio (OR) is greater than 1. (C) GO term enrichments for genes upregulated in MCF10A, MCF10AT1, or MCF10CA1a. Size indicates p-value. Terms are color-coded based on gene type; morphogenesis (purple), proliferation (orange), and cell adhesion (teal). (D) An example of an upregulated gene (COL12A1) with a promoter that overlaps a strengthened loop with distal enhancers. Black boxes show loop annotations, while red boxes indicate diYerential loops. Red compartment tracks indicate A compartments, while blue indicates B compartments. In CTCF signal tracks, red highlights indicate diYerential CTCF peaks. In H3K27ac and ATAC-Seq signal tracks, red highlights indicate diYerential enhancers as determined by changes in H3K27ac. Genes highlighted in black are diYerentially expressed. (E) An example of a downregulated gene (WNT5A) with a promoter that overlaps with several weakened loops containing distal enhancers that lose H3K27ac. Plots are annotated as in (A).

Article Snippet: ChIPseq for CTCF (Cell Signaling Technology, catalog number 3418) and histone marks H3K27ac (Abcam, ab4729) and H3K27Me3 (Abcam ab6002).

Techniques: