Journal: Cancer Discovery

Article Title: Senescent-Like Neutrophils Shape Angiogenic Immunosuppressive Niches in Colorectal Cancer Liver Metastasis

doi: 10.1158/2159-8290.CD-25-0820

Figure Lengend Snippet: Tissue specification of neutrophils in the CRLM microenvironment. A, Expression levels of CXCR4, CXCR2, and CXCR1 across each TAN and PMN subset. B, mIHC staining of MPO, CXCR1, CXCR2, and CXCR4 in colorectal cancer and CRLM tissues. Scale bar, 50µm. C, Quantitative analysis of the percentage of CXCR1 + , CXCR2 + , and CXCR4 + neutrophils in paired colorectal cancer and CRLM tissues ( n = 5). D, Heatmap showing the mean strength of chemokine–receptor axis for each TAN subset in CRLM tissues. E, Predicted ordering of each TAN subset by CytoTRACE analysis using scRNA-seq dataset. F, Monocle 2 trajectory analysis of TAN subsets differentiation using the scRNA-seq dataset. Cells are colored by pseudotime, ordering predicted by CytoTRACE, and cell types. G, The density of each TAN subset along pseudotime. H, Clustering analysis of gene expression along pseudotime, with pathway enrichment analysis of four gene sets based on pseudotime. I, TAN1 similarity scores of TAN0_ELL2, TAN2_HSPA1B, TAN3_IFIT2, and TAN4_ARG1 across colorectal cancer, adjacent liver tissues (LP), and CRLM tissues. The TAN1 similarity score was calculated based on the top 50 differentially expressed genes in TAN1. J, Relative mRNA expression levels of BHLHE40 , CDKN1A (p21), LGALS3 , VEGFA , and LDHA as determined by qPCR in control neutrophils or neutrophils cocultured with HCT116 or DLD-1 cells. K and L, Flow cytometry analysis of Gal3 surface expression in neutrophils cultured alone (control) or cocultured with DLD-1 ( K ) or HCT116 cells ( L ). P values were determined by paired Student t test ( C ) or unpaired Student t test ( J , K , and L ). n.s., not significant; **, P < 0.01; ***, P < 0.001. CRC, colorectal cancer; Ctrl, control; MFI, mean fluorescence intensity.

Article Snippet: The human colon cancer cell lines HCT116 (RRID: CVCL_0291) and DLD-1 (RRID: CVCL_0248), mouse colon cancer cell line CT26 (RRID: CVCL_7254), as well as the human neutrophil cell line HL-60 (RRID: CVCL_0002) were obtained from the American Type Culture Collection.

Techniques: Tissue, Expressing, Staining, Analysis, Gene Expression, Control, Flow Cytometry, Cell Culture, Fluorescence

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , DNA FISH validation of a HU-specific loop in Hap1 cells. Left, representative images of left (green) and right (red) anchor probes (DAPI, blue). Right, the interprobe distance distribution. n = 108 (UT) and n = 119 (HU) S phase nuclei; n = 102 (UT), n = 81 (HU) non-S-phase nuclei. Statistical analysis was performed using two-sided Mann–Whitney U -tests; P = 0.0001 (S phase) and P = 0.4295 (non-S phase). Data were pooled from two independent experiments and normalized to their respective UT conditions (set to 1) to enable direct comparison across experiments. Scale bars, 5 μm. b , Representative HU-specific, G9a-dependent chromatin loops on chromosomes 9 (top) and 2 (bottom). The bottom tracks show H3K9me3 Rep-ChIC peaks, IZs and annotated genes. c , The fractions of IZs and TZs located within HU-unique loop bodies, anchors or neither. d , The aggregate average CTCF Rep-ChIC signal within HU-unique loops and ±10 kb around their anchors. Statistical analysis was performed using the two-sided Mann–Whitney U -test; P = 1.96 × 10 −16 . The results shown are from one biological replicate. e , The distribution of IZs and CTCF motifs within HU-specific loops and flanking regions. Consensus CTCF motifs at loop anchors enriched in the CTCF Rep-ChIC signal and their orientations (convergent versus tandem) are shown below. Heat maps of the CTCF Rep-ChIC signal around loop anchors (±1 kb) in UT and HU-treated MRC5 cells are also shown. f , g , APA of HU-specific loops in HCT116 CTCF-mAID2-mClover cells ( f ) and MRC5 cells ( g ). The enrichment relative to the background is indicated in the top-right corners. Results shown are from one biological replicate. h , Schematic of a loop with CTCF sites (top). Bottom, heat maps of IZs, H3K9me3, FANCD2 Rep-ChIC and the strand-specific fork pausing signal (TrAEL-seq) within HU-unique loops and flanking regions (±3.5 kb). C, Crick strand; W, Watson strand. i , Fork pausing signal within and flanking HU-unique loops with (+CTCF) or without (−CTCF) CBSs at the loop anchor, and UT-HU common loops. Results shown are a representative dataset out of two independent biological replicate. j , The average TrAEL-seq Watson (blue) and Crick (orange) strand profiles across HU-unique loops ±5 kb. All experiments used MRC5 cells unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Biomarker Discovery, MANN-WHITNEY, Comparison

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Distribution of TADs by replication timing (early, late, TTR: Timing Transition Regions) ± HU for two replicates (MRC5 cells). b, c , Insulation score ( b ) and CTCF Rep-ChIC signal ( c ) ± 10 kb of HU-unique loops overlapping (blue/top) or not overlapping (green/bottom) TAD borders. Aggregate average (top) and tornado plots (bottom) are shown. A1 and A2 indicate the loop anchors. Data shown are from one biological replicate. d , Top: CTCF-EdU PLA signal (red) in nuclei (blue). Bottom: Distribution of total PLA spot intensity per nucleus. S-phase nuclei analysed n = 986, 1416, 1556, 1524, 1204 and 846 (left to right) from one representative experiment which has been performed 2 times with similar results. ****P ≤ 0.0001, ns=non-significant (Kruskal–Wallis, Dunn’s test, P values from left to right: <0.0001, <0.0001, <0.0001, 0.3113). Experiments in HCT116-CTCF-mAID2-mClover cells. e , Western blot of CTCF depletion via 5-Ph-IAA, in HCT116-CTCF-mAID2-mClover cells. For gel source data, see Supplementary Fig. [n = 3] independent replicates. f , Heatmaps of average CTCF/FANCD2 Rep-ChIC, Fork-Deg-seq (WT vs. shBRCA2, in RPE1-shBRCA2 cells), γH2AX , and tumour SNVs across HU-unique loops with (+CTCF) or without (-CTCF) CTCF Binding Sites (CBSs) at the anchors for 3 biological replicates. g, h , Heatmaps of IZs (TrAEL-seq MRC5 IZ (g) or HCT116 IZ (h)), H3K9me3, FANCD2 Rep-ChIC, and strand-specific fork pausing signal (W for Watson strand and C for Crick strand, MRC5 cells) within HU-unique loops ± 3.5 kb in MRC5 ( g ) and HCT116-CTCF-mAID2-mClover ( h ) cells. i , Average TrAEL-seq Watson (blue) and Crick (orange) profiles ± 5 kb of HU-unique loops in HCT116-CTCF-mAID2-mClover cells (see Fig. ). All experiments in MRC5 cells unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Insulation, Western Blot, Binding Assay

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Rolling mean of z-scored DAPI intensity vs. S-phase progression from scEdU-seq tracks. Ribbon indicates s.d. b , Number of forks per cell vs. S-phase progression. Line is median; ribbon is 95% CI. c , Heatmap of maximum normalized scEdU-seq log counts binned per 40 kb along a 60 Mb region of chromosome 2, ordered by S-phase progression. Colour scale: Normalized read coverage across the region. d, e , DNA replication speeds at indicated loop anchors and background regions. Number of regions analysed: d, [n = 345 and 192 for UT and 0.5mMHU respectively] and e, n = 302, 283, 302, 214, 203 and 215 (left to right). Results shown are from one representative experiment which has been performed 2 times with similar results. f, g , Schematic of DNA fibre degradation assay in HCT116-CTCF-mAID2-mClover (top). IdU/EdU track length ratio distributions. Means ± s.d. shown (bottom). Number of tracks analysed: Panel f: n = 1015, 1058, 1057, 1057, 1116, 1080, 1117, 1079, 1101, 1086, 1069 and 1061 (left to right), pooled from 3 independent biological replicates and overlaid in three different colours in the plot. Panel g: n = 681, 726, 708, 724, 641, 669, 643, 676, 682, 684, 683, 666, 703, 676, 676, 689, 673, 686, 713 and 665 (left to right) pooled from 2 independent biological replicates and overlaid in two different colours. ****P ≤ 0.0001, **P ≤ 0.01, ns=non-significant (Kruskal–Wallis, Dunn’s test, panel f: All P values < 0.0001, panel g: P values: (left to right) >0.9999, <0.0001, <0.0001, <0.0001, <0.0001, 0.0095, 0.0061, <0.0001, >0.9999, <0.0001, 0.4485, >0.9999, 0.0019, >0.9999, >0.9999, >0.9999). h , Schematic of Fork-Deg-seq approach to map nucleolytic degradation at newly replicated regions. The diagram was created using BioRender; Taneja, N. https://BioRender.com/d7kf3t0 (2026). i , Fork-Deg-seq signal in WT and shBRCA2-induced RPE1 cells after 8 h 4 mM HU, alongside BrdU IP signal and replication timing for the indicated region on chromosome 3. Dotted square highlights a loop-poor region with enhanced Fork-Deg-seq enrichment. Loops of bidirectional replicon (fountain)- scale are shown in dark red; smaller loops are shown in light red. j , Aggregate heatmap of CTCF (CTCF-Rep-ChIC, in MRC5 cells), ForkDeg-seq signal (in RPE1-shBRCA2 cells), γH2AX , and Single Nucleotide Variant (SNV ) distribution within and in a +/− 1 kb region flanking the HU-specific loops identified in MRC5 cells. Schematics: loop body and flanking regions. Orange triangles mark loop anchors position based on CTCF-binding sites. Colour scale: Normalized read coverage across the region. All experiments in MRC5 cells unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Degradation Assay, Variant Assay, Binding Assay

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Schematic of the replication fork degradation DNA fibre assay in HCT116 CTCF-mAID2-mClover cells, involving CTCF depletion (dep; 5-Ph-IAA) and G9a inhibition (UNC0642) (top). Middle, representative fibres. Bottom, the IdU/EdU track length ratio. Data are mean ± s.d. From left to right, numbers of forks analysed per condition: n = 1,014, 1,015, 1,034, 1,039, 1,032, 1,053, 1,070 and 1,005, pooled from three independent replicates and overlaid in three different colours in the plot. Statistical analysis was performed using Kruskal–Wallis tests followed by Dunn’s test; from left to right, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P > 0.9999, P > 0.9999, P > 0.9999. Scale bar, 5 μm. b , Representative locus (chromosome 16: 81.3–82.75 Mb). Top, Hi-C heat map (the red squares highlight the positions of the loop anchors). The Fork-deg-seq signal in HCT116-CTCF-mAID2-mClover cells UT or treated with G9ai (4 h), 5-Ph-IAA (4 h, CTCF-depleted) or both after 4 mM HU (5 h or 8 h). MRC5 CTCF and H3K9me3 Rep-ChIC signals are shown below, alongside IZs and fragile sites. The black arrowheads indicate high Fork-deg-seq signal. The shaded area shows a loop-dense region with reduced degradation; unshaded areas show enhanced Fork-deg-seq enrichment. c , BrdU-enriched 5-kb bins classified by HU-unique loop coverage: loop-poor (0–1 loop, left, n = 4,398 bins) and loop-dense (≥2 loops, right, n = 6,844 bins). The fold change in Fork-deg-seq signal relative to the WT is shown. Data are mean ± s.d. Statistical analysis was performed using two-sided Mann–Whitney U -tests; loop-free region, from top to bottom: P = 4.4 × 10 −33 , P = 2.4 × 10 −132 , P = 2.4 × 10 −132 , P = 4.1 × 10 −33 , P = 2.4 × 10 −132 , P = 2.7 × 10 −34 ; loop-dense region, from left to right: P = 1.4 × 10 −130 , P = 2.0 × 10 −130 , P = 1.8 × 10 −130 , P = 3.2 × 10 −1 , P = 7.8 × 10 −2 , P = 8.4 × 10 −1 . Results shown are from one biological replicate. d , Aggregate analysis of the mean ± s.d. Fork-deg-seq signal within HU-unique loops and 5 kb flanking regions after 3 h of treatment with 4 mM HU alone (top row, left four plots) or with mirin and DNA2i followed by 4 mM HU (bottom row). Ionizing radiation (10 Gy) was included as a control without further treatment (top right plot). The results shown are from one biological replicate. All of the experiments described in this figure were performed in HCT116 CTCF-mAID2-mClover cells, unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Inhibition, Hi-C, MANN-WHITNEY, Control

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Representative locus (chr8: 122.8-133.9 Mb). Top: Hi-C heatmap (red squares highlight positions of loop anchors). Below: Fork-Deg-seq signal (HCT116-CTCF-mAID2-mClover cells) untreated or treated with G9ai (4 h), 5-Ph-IAA (4 h, CTCF-dep) or both upon 4 mM HU (5 h or 8 h), alongside MRC5 CTCF and H3K9me3 Rep-ChIC signals, IZs and fragile sites. Black arrowheads indicate high Fork-Deg-seq signal. Shaded area: loop-dense region with reduced degradation; unshaded areas show increased degradation. b , Aggregate mean Fork-Deg-seq signal ± s.d. after 5 h 4 mM HU within HU-unique loops ± 5 kb in HCT116-CTCF-mAID2-mClover cells. Results shown are from one representative experiment which has been performed 2 times with similar results. c-e , Distribution of IZs at early [n = 2119] and late [n = 2193] replicating fragile sites. ( c ) HU-unique loops overlapping IZs (MRC5), ****P ≤ 0.0001, (Two-sided Mann–Whitney U test, p-value: 7.858e-38). ( d ) Fork-Deg-seq signal in HCT116 IZs, ****P ≤ 0.0001, (Two-sided Mann–Whitney U test, p-values: 1.222e-07, 6.247e-06, 1.161e-05, 6.247e-06 left to right). ( e ) Fork-Deg-seq signal in RPE1 IZs (RPE1-shBRCA2 cells), ****P ≤ 0.0001, (Two-sided Mann–Whitney U test, p-values: 3.119e-37 WT + HU and 2.080e-55 shBRCA2+HU). f , Representative electron micrographs showing a reversed fork with ssDNA gaps on both daughter strands and intact reversed arms (HCT116-CTCF-mAID2-mClover). P, parental strand; D, daughter strand; R, reversed arms. Scale bars: 250 nm or 1183 bp (main), 50 nm or 473 bp (insets). g , Ranked gap length distribution per condition. h , qPCR analysis of HU-unique loop formation in WT and TKO (SMARCAL1, ZRANB3, HLTF knockout) U2OS cells ± 4 mM HU. Means ± s.e.m. [n = 4 independent biological replicates]. ****P < 0.0001, ***P < 0.001, **P < 0.01, ns=non-significant (Ordinary one-way ANOVA, Tukey’s test, P value from top to bottom: a: <0.0001, 0.0026, 0.4735; b: 0.0003, 0.0095, 0.5794; c: 0.004, 0.1237, 0.3688; d: <0.0001, 0.0471, 0.1211; e: <0.0001, 0.0003, 0.0752; f: 0.0004, 0.0003, 0.5844; g: <0.0001, 0.0042, 0.4301; h: 0.0003, 0.0004, 0.935; i: <0.0001, 0.0019, 0.4734,; j: 0.0006, 0.0001, 0.6204; k: 0.0005, 0.0206, 0.4417; l: <0.0001, 0.0032, 0.3017; m: <0.0001, 0.0173, 0.269; n: 0.0013, 0.0074, 0.8887; o: 0.0013, 0.0074, 0.7701; p: 0.0003, 0.095, 0.1693). i , Top: Schematic showing that only H3K9me3 signal intensity overlapping with EdU was measured. Bottom: Distribution of H3K9me3 intensity in the region of interest and for the indicated conditions. Number of replication sites analysed per condition across two independent experiments: n = 148, 98, 111, 107, 103 and 84 from left to right. (*: p < 0.05, ****: p < 0.0001, ns: non-significant. Kruskal–Wallis test followed by Dunn’s test, P values: 0.0160, <0.0001, <0.0001, >0.9999, 0.7905, >0.9999, 0.0946, >0.9999 and 0.0156 (top to bottom)). j , Top: Schematic showing that only the H3K9me3 signal intensity just outside of the EdU track was measure (H3K9me3 intensity over a region covering 20% of the total length of the EdU track was measured on both side of the EdU track). Bottom: Distribution of H3K9me3 intensity in the region of interest and for the indicated conditions. Number of replication sites analysed per condition across two independent experiments: n = 98, 119, 107 and 103 from left to right. (***: p < 0.001, ****: p < 0.0001, ns: non-significant. Kruskal–Wallis test followed by Dunn’s test, P values: <0.0001, 0.7726, 0.4606, 0.0003, <0.0001 and <0.0001 (top to bottom). k , Total intensity distribution of CTCF-EdU PLA spots. S-phase nuclei analysed: n = 642 for all conditions imaged from one representative experiment, which has been performed 2 times with similar results. Red line marks mean. ****P ≤ 0.0001, **P ≤ 0.01, ns=non-significant (Kruskal–Wallis, Dunn’s test, P values from left to right: <0.0001, <0.0001, 0.0011, <0.0001, <0.0001, 0.1690).

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Hi-C, MANN-WHITNEY, Knock-Out

Journal: Aging and Disease

Article Title: Postbiotic Parabacteroides Distasonis Supplementation Enhances Intestinal and Skeletal Muscle Function in Aged Mice

doi: 10.14336/AD.2025.0188

Figure Lengend Snippet: Postbiotic Pd modifies mitochondria in a colon cell line. (A) Right panel-representative images of HCT116 treated or not with postbiotic Pd for 24h and labeled with TMRE to determine mitochondrial morphology. Bar = 10 µm. Left panel- bar graphs of the mitochondrial length analysis. Data are expressed as MEAN ± SEM of five independent experiments ****p≤0.0001. Mann-Whitney test . (B) Upper panel- representative Western blot of PGC1α and GADPH as a loading control in HCT116 cells treated or not with postbiotic Pd. Bottom panel- Bar graphs represent quantification of PGC1α/GADPH expressed as MEAN ± SEM of five independent experiments. ∗p < 0.05. Mann-Whitney test . (C) Upper panel- representative Seahorse trace of HCT116 cells treated or not with postbiotic Pd. A; oligomycin (1 µM), B;FCCP (250 µM), C; rotenone plus antimycin A (1 µM each). Bottom panel- basal and maximum OCR of HCT116 cells treated or not with postbiotic Pd. MEAN ± SEM of three independent experiments with 10 replicates each. ***P < 0.001 compared to control. Mann-Whitney test. (D) Upper panel- representative Western blot of CHOP and β-actin as a loading control in HCT116 cells treated or not with postbiotic Pd. Bottom panel- bar graphs represent quantification of CHOP/β-actin expressed as MEAN ± SEM of 3 independent experiments. ∗p < 0.05. Mann-Whitney test . (E) Upper panel- representative Western blot of VDAC1 and β-actin as a loading control in colon mucosa samples from 26-months-old mice treated or not with postbiotic Pd. Bottom panel- bar graphs represent quantification of VDAC1/β-actin expressed as MEAN ± SEM. Note that β-actin blot in is the same as in this figure, as the same membrane was stripped and re-probed for VDAC1. N = 3 in the control group and 4 for the Pd treated group. ∗p < 0.05. Mann-Whitney test .

Article Snippet: Human colorectal cell line HCT116 (ATCC) was maintained at 37oC (95%/5% air/CO 2 ) in DMEM media (GIBCO) supplemented with 10% (v/v) FBS.

Techniques: Labeling, MANN-WHITNEY, Western Blot, Control, Membrane