Journal: iScience

Article Title: CTCF/cohesin-binding sites are susceptible to replication-associated DNA damage and genomic instability in cancer cells

doi: 10.1016/j.isci.2026.114646

Figure Lengend Snippet: CTCF and cohesin remain co-bound to DNA in the replication phase (A) HeLa cells were synchronized to various phases of the cell cycle by using thymidine arrest/release or nocodazole block method (see STAR Methods). The cell cycle profile in each stage was confirmed by staining with propidium iodide (PI) and analyzed on a BD FACSVerse flow cytometer. In each histogram, cell count was plotted against DNA content measured from the PI fluorescence signal. The percentage of cells in each phase of the cell cycle is mentioned in brackets. (B) The chromatin-bound fraction of CTCF and RAD21 at various cell cycle phases of HeLa cells was checked by western blotting. GAPDH and Histone H3 were used as loading control for cytoplasmic and chromatin-bound fractions, respectively. (C) Representative FRAP images of GFP-tagged CTCF in asynchronous (top panel) and Mid S (bottom panel) HeLa cells. The scale bar is 5 μm. (D) Fluorescence recovery curve of GFP-tagged CTCF for 3 min post bleaching in asynchronous and Mid S HeLa cells (37 and 41 cells in asynchronous and S phase, respectively) from three biological replicates. The normalized intensity value is plotted on the y axis (each dot in the graph represents mean±1SD). (E) Venn diagram showing the overlapping peaks of CTCF and RAD21 in Mid S HeLa cells, and the number of high-confident CTCF/cohesin-binding sites (CBSs) with presence of CTCF motif (predicted within the CTCF peak) and RAD21 peak within 100bp from the CTCF motif (see ). (F) An example genome browser snapshot of a region on chr19 showing the overlap between ChIP-seq peaks in asynchronous and Mid S cells. From top to bottom: ChIP-seq signal in CTCF asynchronous, CTCF Mid S (two replicates), RAD21 asynchronous (from ENCODE), and RAD21 Mid S (two replicates) in HeLa cells. (G) Overlap of CBSs in the Mid S phase HeLa cells with CBSs defined from various cell lines in asynchronous condition (from ENCODE). The number of CBSs identified in each cell line is mentioned in brackets.

Article Snippet: Antibodies used are: CTCF (3418: Cell Signaling Technology, 1 μg), RAD21 (ab992: Abcam, 1 μg), MRE11 (ab208020: Abcam, 2 μg), γH2AX (ab81299: Abcam, 2 μg), H2AX (ab11175: Abcam, 2 μg), RAD51 (ab176458: Abcam, 2 μg), ATM (ab201022: Abcam, 2 μg) and FANCD2 (NB100-182: Novus Biologicals, 2 μg).

Techniques: Blocking Assay, Staining, Flow Cytometry, Cell Characterization, Fluorescence, Western Blot, Control, Binding Assay, ChIP-sequencing

Journal: iScience

Article Title: CTCF/cohesin-binding sites are susceptible to replication-associated DNA damage and genomic instability in cancer cells

doi: 10.1016/j.isci.2026.114646

Figure Lengend Snippet: Enrichment of replication stress-associated proteins at CTCF/cohesin-binding sites (A) ChIP-seq signal profile of MRE11 in Mid S HeLa cells plotted across a ±5 kb window centered at CBSs from Mid S HeLa (maroon), CTCF unbound sites (sky blue), and random genomic regions (yellow). A total of 26,948 sites were in each category. The heatmap (on the right) represents the ChIP-seq signal for each site, and the profile plot (on the left) represents the average signal across all sites. (B) Similarly, the ChIP-seq profile of STN1 in Mid S upon HU treatment (from Chastain et al. ) plotted surrounding CBSs and control sites (±5 kb). (C and D) ChIP-seq signals of (C) MRE11 and (D) STN1 (+HU) in Mid S phase plotted surrounding CBSs (maroon; 26,948 sites), CTCF-alone sites (gray; 24,636 sites), and RAD21-alone sites (green; 9,135 sites). (E) MRE11 profile at CBSs based on CTCF and RAD21 binding strength. Light red: CBSs with both CTCF and RAD21 low-binding strength (4,694 sites). Dark red: CBSs with both CTCF and RAD21 high-binding strength (4,775 sites). (F) STN1(+HU) occupancy in the Mid S phase at CTCF/RAD21 low- or high-binding CBSs spanning a ±5 kb window.

Article Snippet: Antibodies used are: CTCF (3418: Cell Signaling Technology, 1 μg), RAD21 (ab992: Abcam, 1 μg), MRE11 (ab208020: Abcam, 2 μg), γH2AX (ab81299: Abcam, 2 μg), H2AX (ab11175: Abcam, 2 μg), RAD51 (ab176458: Abcam, 2 μg), ATM (ab201022: Abcam, 2 μg) and FANCD2 (NB100-182: Novus Biologicals, 2 μg).

Techniques: Binding Assay, ChIP-sequencing, Control

Journal: iScience

Article Title: CTCF/cohesin-binding sites are susceptible to replication-associated DNA damage and genomic instability in cancer cells

doi: 10.1016/j.isci.2026.114646

Figure Lengend Snippet: Enrichment of DNA damage response and repair proteins at CBSs (A and B) ChIP-seq signal profile and heatmap of (A) γH2AX (normalized with H2AX) and (B) RAD51 from Mid S HeLa cells plotted at CBSs (26,948 sites), CTCF unbound (26,948 sites), and random regions (26,948 sites). (C and D) ChIP-seq signal of (C) γH2AX (normalized with H2AX) and (D) RAD51 plotted at CBS Mid S (26,948 sites), CTCF-alone (24,636 sites), and RAD21-alone (9,135 sites). (E and F) ChIP-seq signal of (E) γH2AX (normalized with H2AX) and (F) RAD51 at CBSs with low- (4,694 sites) and high-binding strength of CTCF/RAD21 (4,775 sites). For the γH2AX signal ±10 kb flanks were considered, while RAD51 signal is plotted at ±5 kb regions.

Article Snippet: Antibodies used are: CTCF (3418: Cell Signaling Technology, 1 μg), RAD21 (ab992: Abcam, 1 μg), MRE11 (ab208020: Abcam, 2 μg), γH2AX (ab81299: Abcam, 2 μg), H2AX (ab11175: Abcam, 2 μg), RAD51 (ab176458: Abcam, 2 μg), ATM (ab201022: Abcam, 2 μg) and FANCD2 (NB100-182: Novus Biologicals, 2 μg).

Techniques: ChIP-sequencing, Binding Assay

Journal: iScience

Article Title: CTCF/cohesin-binding sites are susceptible to replication-associated DNA damage and genomic instability in cancer cells

doi: 10.1016/j.isci.2026.114646

Figure Lengend Snippet: Enrichment of somatic mutations at CBSs in STN1/MRE11-deleted tumors (A and B) Mutation rate plotted at the ±1 kb region of CBSs Mid S and control sites (CTCF unbound, CTCF-alone, and RAD21-alone sites) in stomach adenocarcinoma samples with (A) both MRE11 and STN1 wild type (No. of samples, N = 38) and (B) both MRE11 and STN1 deletion (No. of samples, N = 4). p -values were calculated by using G-test. (C) Mutation fold change at CBSs ( ±20 bp) core in tumor samples with either MRE11 or STN1 deletion (or both deletion) relative to mutation fold change in MRE11 and STN1 wild-type samples across different tumor types. The statistical difference was calculated by using the Fisher exact test. p -value annotation legend ∗∗: 0.001 < p ≤ 0.01, ∗∗∗∗: p < 0.0001. (D) Mutation fold change at CBSs ( ±20 bp) core stratified based on the CTCF and RAD21 binding strength. On the x axis, the high binding represents CBSs with both CTCF and RAD21 strong binding and low binding represents CBSs with both CTCF and RAD21 weak binding. The statistical difference was calculated by using the Fisher exact test. p -value annotation legend ∗: 0.01 < p ≤ 0.05, ∗∗: 0.001 < p ≤ 0.01, ∗∗∗: 0.0001 < p ≤ 0.001, ∗∗∗∗: p < 0.0001.

Article Snippet: Antibodies used are: CTCF (3418: Cell Signaling Technology, 1 μg), RAD21 (ab992: Abcam, 1 μg), MRE11 (ab208020: Abcam, 2 μg), γH2AX (ab81299: Abcam, 2 μg), H2AX (ab11175: Abcam, 2 μg), RAD51 (ab176458: Abcam, 2 μg), ATM (ab201022: Abcam, 2 μg) and FANCD2 (NB100-182: Novus Biologicals, 2 μg).

Techniques: Mutagenesis, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Clostridioides difficile TcdB induces expression of its receptor (CSPG4) through a noncanonical Hippo signaling mechanism

doi: 10.1016/j.jbc.2026.111137

Figure Lengend Snippet: CSPG4 is regulated by CTCF . A , heatmaps representing Z-score values for the top TcdB induced genes that significantly differ from the TcdB + XMU-MP-1 group. These data are obtained from RNA-seq analysis of pericytes exposed for 24 h to 1 ng/ml of TcdB and/or 10 μM XMU-MP-1. B , representative bands from a capillary separation immunodetection system obtained from protein lysates generated from HeLa cells and HeLa CTCF-KD . C , genome browser tracks displaying CUT&RUN experiment for HeLa cells left untreated or exposed to 1 ng/ml of TcdB2 for 24 h. D , heatmaps displaying CTCF changes in DNA binding throughout the genome. E , model describing how TcdB modulates Hippo signaling and increases CSPG4.

Article Snippet: Primary antibodies used were a mouse monoclonal antibody recognizing nonglucosylated Rac1 (BD Bioscience; catalog no. 610651); a mouse monoclonal antibody recognizing total Rac1 (EMD Millipore; catalog no. 05–389); a mouse monoclonal antibody against GAPDH (Abcam; product # ab8245); a mouse monoclonal against YAP/TAZ (Santa Cruz Biotechnology; product # sc-101199); a mouse monoclonal against MST1 (Santa Cruz Biotechnology; product # sc-515051); a rabbit monoclonal antibody against CSPG4 (Abcam; ab275024); a rabbit monoclonal antibody against ß-actin (Cell Signaling Technology; product # 4970); a rabbit monoclonal antibody against CTCF (Cell Signaling Technology; product # 3418); a rabbit monoclonal against LATS1 (Cell Signaling Technology; product #3477) a rabbit monoclonal against phosphorylated LATS1 (Thr1079) (Cell Signaling Technology; product #8654); and a rabbit monoclonal antibody against CTGF (Cell Signaling Technology; product # 86641).

Techniques: RNA Sequencing, Immunodetection, Generated, Binding Assay