Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured in low or high density. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without serum. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without aphidicolin. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. cGAS in nuclear/cytosolic fractions of indicated cell types. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Western Blot, Cell Culture

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Fluorescence images of GFP‐hcGAS, GFP‐hcGASΔcGAMP, GFP‐hcGASΔDNA, and GFP‐hcGASΔOligo in HEK293 cells cultured with or without aphidicolin. Scale bar: 20 μm. B Corresponding quantification of (A). The nuclear cGAS/total cGAS was calculated from 6 different fields with n > 50 cells. C, D A nuclear export signaling (NES) is not sufficient to dislodge chromatin‐bound cGAS from the nucleus. (C) Fluorescence images of GFP‐hcGAS, GFP‐hcGAS‐NLS, and GFP‐hcGAS‐NES in HEK293 cells. Scale bar: 10 μm. (D) Immunoblots of subcellular fractions of GFP‐hCGAS‐, GFP‐hCGAS‐NLS‐, and GFP‐hCGAS‐NES‐expressing HEK293 cells. Data information: Data are presented as means ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS: P > 0.05 and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Fluorescence, Cell Culture, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A, B Micronuclei (indicated by arrowhead) in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells before (0 h) or 24 h after γ‐irradiation (IR; 10 Gy). Scale bar: 10 μm (A). (B) The average MNs/cell. Graphs show mean ± SEM ( n = 3 independent experiments) representing six different microscopic fields with over 200 cells. C IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n = 3 independent experiments. D Experimental outline for micronucleus generation and cell death after γ‐irradiation. E Micronucleus (indicated by arrowhead) and cGAS staining in WT and cGAS −/− BMDMos exposed to γ‐irradiation (10 Gy). Scale bar: 10 μm. F Average MNs/cell in BMDMos. MN graphs show mean ± SEM ( n = 3 independent experiments) representing eight different microscopic fields with over 200 cells. G Cell death in WT and cGAS −/− BMDMos that were first synchronized at G2/M, then γ‐irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ± SD, x biological triplicates ( n = 3) per treatment group are shown. Data information: Statistical significance in (B), (C), and (F) was assessed using unpaired two‐tailed Student's t ‐test. *** P ≤ 0.001 and **** P ≤ 0.0001. Statistical significance in (G) was assessed using two‐way ANOVA test, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Irradiation, Transfection, Plasmid Preparation, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Pulsed‐field gel electrophoresis analysis of γ‐irradiated (10 Gy) WT and cGAS −/− BMDMos. B, C Comet assay in GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells γ‐irradiated (IR: 10 Gy) for 15 min (B). RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293T cells stimulated with transfected DNA for 6 h (C). D, E Comet assay of HEK293 cells stimulated with 10 μg/ml cGAMP for indicate periods, then γ‐irradiated and incubated at 37°C for indicated duration (D). (E) Immunoblots of IRF3 phosphorylation in HEK293 cells treated as in (D). F–H Images (F) and quantifications (G) of comet tails 15 min after irradiation of GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells. RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells stimulated with transfected 23 DNA for 6 h (H). I, J Images (I) and quantifications (J) of micronuclei in GFP‐NLS‐ and GFP‐hcGASΔcGAMP‐expressing HEK293 cells 24 h after γ‐irradiation (IR; 10 Gy). DAPI (DNA). Scale bar: 10 μm. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. K Quantifications of comet tails 15 min after irradiation (10 Gy) of GFP‐NLS‐, GFP‐hcGAS‐, or GFP‐mcGAS‐expressing HEK293 cells. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. Data information: Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS P > 0.05, *** P ≤ 0.001, and **** P ≤ 0.0001. Mean ± SEM of n = 3 independent experiments. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Pulsed-Field Gel, Electrophoresis, Irradiation, Single Cell Gel Electrophoresis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Incubation, Western Blot, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Reporter assays showing the effect of NLS and NES on cGAS‐mediated inhibition of DNA repair. B Both full‐length hcGAS and hcGAS cat (161–522aa) inhibit HR repair. C–E cGAS does not impede ATM activation. ATM phosphorylation in γ‐irradiated (10 Gy) GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells (C), GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells (D), or γ‐irradiated (2.5 Gy) WT, cGAS −/− , and Sting −/− BMDMos (E). Data information: Data are means ± SD, n = 3. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. *** P < 0.001 **** P < 0.0001, NS: P > 0.05. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Irradiation, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A cGAS is not recruited to DSB sites: Confocal microscopic images of GFP‐NLS‐ or GFP‐hcGAS‐expressing U2OS‐DSB reporter cells incubated (or not) with Shield‐1 and 4‐OHT to induce the expression and translocation of mCherry‐LacI‐FokI (red) to specific DSB sites. Scale bar: 10 μm. The arrowheads indicate DSB sites. B cGAS does not co‐localize with γ‐H2AX at DSB sites: GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells exposed (or not) to γ‐irradiation (IR: 10 Gy), then stained for γ‐H2AX. Scale bar: 10 μm. C, D Nuclear cGAS is mainly chromatin‐bound and remains unaltered upon γ‐irradiation. (C) Cytosolic (cyto) and nuclear fractions of γ‐irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules. (D) Cytosolic, soluble nuclear, and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins. E–G cGAS co‐isolates with DNA repair proteins because of bound chromatin bridges. (E) Nuclease digestion abrogates the co‐isolation of cGAS and DNA repair proteins: Lysates of control (−IR) and γ‐irradiated (+IR, 10 Gy, 30 min) GFP‐hcGAS‐expressing HEK293 cells were treated (or not) with benzonase before cGAS immunoprecipitation and analysis for indicated proteins. (F) Agarose gel analysis of DNA in corresponding cell lysates in (E). (G) Co‐isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ‐irradiated HEK293 cells expressing GFP‐hcGAS or GFP‐hcGASΔDNA probed for indicated proteins. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Incubation, Translocation Assay, Irradiation, Staining, Isolation, Control, Immunoprecipitation, Agarose Gel Electrophoresis, Binding Assay

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Confocal images of γ‐irradiated GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293 cells stained for RAD51 (red) with or without γ‐irradiation. Scale bar: 10 μm. Schematics of the D‐loop formation assay, including pre‐incubation of template dsDNA with cGAS cat (i) or with cGAS cat being added after RAD51 was bound to dsDNA (ii). Pre‐incubation of dsDNA with mcGAS cat prevents D‐loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D‐loop formed in each reaction (left) was graphed as the average of triplicates ± SD. Schematics of the D‐loop assay. Pre‐incubation of template dsDNA with hcGAS cat blocks subsequent D‐loop formation. The percentage of D‐loop formation (below) was graphed as the average of triplicates ± SD. Data information: Unpaired two‐tailed Student's t ‐test was used for statistical analyses. NS P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Irradiation, Expressing, Staining, Tube Formation Assay, Incubation, Activity Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Negative‐stain electron micrographs of cGAS‐dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation when pre‐incubated with dsDNA. Percentage of D‐loop formed in each reaction (left) graphed as the average of triplicates ± SD. Overview of a single 1:1 hcGAS‐DNA complex depicting the location of the Y215 within the cGAS‐dsDNA interface. DR‐GFP assay showing that hcGASΔDNA‐Y215E is impaired in HR inhibition. hcGASΔDNA‐Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24. hcGASΔDNA‐Y215E and hcGASΔcGAMP are defective in synthase activity. Negative‐stain electron micrographs showing that hcGAScat‐ΔDNA‐Y215E is defective in inducing cGAS‐dsDNA complexes. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation. Data information: Data are means ± SD, n = 3. Unpaired Student's t ‐test was used for statistical analyses: NS P > 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Staining, Incubation, Inhibition, Activity Assay

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Distribution of CD44 + cells within intestinal epithelium. a , b Immunohistochemical (IHC) staining for Lgr5 + ISCs ( black dotted lines ) in vivo. c , d IHC staining for CD44 + cells ( black dotted lines ) in vivo. a , c Magnification ×400. Bars 50 μm. b , d Magnification ×1000. Bars 20 μm. e–n Immunocytochemical (ICC) staining for CD44 + cells in vitro. e , j Differential interference contrast (DIC) imaging. f , k Propidium iodide (PI) staining for nuclei. g , l Fluorescein isothiocyanate (FITC) for CD44 + cells ( white arrowheads crypt cells strongly positive for CD44). h , m Overlay of PI image and FITC image. i , n Overlay of FITC image and DIC image. e–i Magnification ×200. Bars 200 μm. j–n Magnification ×630. Bars 100 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Immunohistochemical staining, Immunohistochemistry, In Vivo, Staining, In Vitro, Imaging

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Identification of CD44 + cells. a–a’’’’’ Fluorescence-activated cell sorting (FACS) analysis for cellular phenotype. a Isotype control, IgG2a-phycoerythrin (PE). a’ CD31-PE. a’’ CD34-PE. a’’’ Isotype control, IgG2b-allophycocyanin (APC). a’’’’ CD44-APC ( lo low-positive for CD44, hi high-positive for CD44). a’’’’’ CD45-APC. b Development of CD44 + ISC in 3D-culture system. Numbers represent days. Top Magnification ×400. Bar 50 μm. Bottom Magnification ×200. Bars 100 μm. c–c’’ Colony-forming efficacies of CD44 - cells and CD44 + cells. c CD44 - cells in 3D-culture system for 14 days. c’ CD44 + cells in 3D-culture system for 14 days. c , c’ Magnification ×40. Bars 500 μm. c’’ Comparision of colony-forming efficacy per 100 sorted cells seeded in one well of a 96-well plate. CD44 - group in 48 wells; CD44 + group in 48 wells. Data represent means ± SD of 48 independent measurements ( n = 48). Bars SD values. Paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 + group versus CD44 - group). All experimental procedures were repeated twice. d–d’’’’ Transmission electron microscope imaging of CD44 + ISC differentiation. CD44 + ISC were cultured in the 3D-system for 6 days and formed a cystic structure. d Cystic structure of a single CD44 + ISC-derived organoid at 6 days. Boxed areas are shown at higher magnification in d’–d’’’’ ( Lu lumen). Magnification ×400. Bar 50 μm. d’ Absorptive cell ( Ab ). d’’ Endocrine cell ( En ). d’’’ Goblet cell ( Go ). d’’’’ Paneth cell ( Pa ). Black arrowhead in d’ indicates brush border. Black arrowheads in d’’–d’’’’ indicates granules. d’–d’’’’ Magnification ×1500. Bars 5 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Fluorescence, FACS, Control, Transmission Assay, Microscopy, Imaging, Cell Culture, Derivative Assay

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: CD44 + ISCs resemble CBC stem cells. a–a’’ Strategy for sorting CD44 - , CD44 low+ and CD44 hi+ subpopulations by using the FACS technique ( R1 determining the cell-zone, R2 determining viable cells, PI propidium iodide, APC allophycocyanin). b Semi-quantitative reverse transcription (RT) followed by the polymerase chain reaction (PCR) for ISC-related gene-expression in sorted cells. Fold expression values were normalized to the CD44 - group. Data represent means ± SD of six independent measurements ( n = 6). Bars indicate value of SD. The paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 hi+ group versus CD44 low+ group); $ P ≤ 0.05 represents low significance (CD44 hi+ group versus CD44 low+ group); ns represents no statistic differences between the CD44 hi+ group and CD44 low+ group. P -values for Lgr5 , Bmi1 , Hopx , mTERT , Ascl2 , Smoc2 , Lrig1 , Rnf43 and Prominin-1 are respectively 0.016, 0.036, 0.010, 0.844, 0.007, 0.001, 0.041, 0.005 and 0.859. c , c’ Colony-forming efficacy of CD44 low+ and CD44 hi+ cells in 3D-culture system for 14 days. c CD44 low+ group. c’ CD44 hi+ group. Magnification ×40. Bars 500 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Expressing

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Epithelial homeostasis in CD44 + ISC-derived organoid. a , b TUNEL staining for apoptotic cells in normal epithelium. c , d IHC staining of Ki67 for proliferative cells within normal epithelium. a , c Magnification ×200. Bars 100 μm. b , d Magnification ×400. Bars 50 μm. e–j Epithelial homeostasis in vitro. e–g TUNEL staining for apoptotic cells in CD44 + ISC-derived organoid. e DAPI staining ( blue ) for nuclei. f dUTP-FITC ( green ) for apoptotic cells. g DAPI image merged with dUTP-FITC image. Magnification ×100. Bars 200 μm. h–j ICC staining for proliferative cells in CD44 + ISC-derived organoid. h DAPI staining for nuclei. i Ki67-FITC for proliferative cells. j DAPI image merged with Ki67-FITC image. Magnification ×200. Bar 100 μm. k Representation of an organoid

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, In Vitro

Journal: Cell reports

Article Title: ΔNp63 drives dysplastic alveolar remodeling and restricts epithelial plasticity upon severe lung injury

doi: 10.1016/j.celrep.2022.111805

Figure Lengend Snippet:

Article Snippet: PolyA-selected RNA was used to generate libraries using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions.

Techniques: Virus, Recombinant, Lysis, Magnetic Beads, Migration, Single-cell Transcriptomics, Software

Journal: Cell Reports Medicine

Article Title: Leucine restriction ameliorates Fusobacterium nucleatum- driven malignant progression and radioresistance in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2024.101753

Figure Lengend Snippet: Intracellular F. nucleatum promotes radioresistance in NPC cells by suppressing host apoptosis and DNA damage (A–G) Fn-infected and uninfected NPC cells were exposed to 2, 4, and 8 Gy irradiation, respectively. (A) Representative images of NPC cells. Fn (MOI = 10:1) or E. coli -infected NPC cells (MOI = 1:100). Scale bar: 150 μm. (B) Cellular viability with live/dead assay. Statistical results are presented in the below panels. Data are mean values of three biology repeats. Scale bar: 100 μm. (C) Representative photographs of colony formation assays. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (D) The apoptosis rates were determined by flow cytometry. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (E) LDH activity in supernatant was assessed by LDH Cytotoxicity Assay Kit; optical density (OD) values of 490 nm were present with histogram. (F) Representative images of the comet assay. Statistical results are presented in the right panels. Data are mean values of three biology repeats. (G) Western blot analysis of γH2AX was performed. Statistical results are presented in the right panels. Data are mean values of three biology repeats. Data are shown as mean ± SD. p values were determined by independent sample t tests (C–E and G), ∗ p < 0.05, ∗∗ p < 0.001, and ∗∗∗ p < 0.001.

Article Snippet: E. coli DH5α , TIANGEN , Cat# CB101-02.

Techniques: Infection, Irradiation, Live Dead Assay, Flow Cytometry, Activity Assay, LDH Cytotoxicity Assay, Single Cell Gel Electrophoresis, Western Blot

Journal: Cell Reports Medicine

Article Title: Leucine restriction ameliorates Fusobacterium nucleatum- driven malignant progression and radioresistance in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2024.101753

Figure Lengend Snippet:

Article Snippet: E. coli DH5α , TIANGEN , Cat# CB101-02.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Viability Assay, ROS Assay, LDH Cytotoxicity Assay, ATP Assay, Sequencing, Virus, Synthesized, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Dual regulation of CXCR6+CD8+ T cells modulates cytotoxic and exhaustion-associated programs during prostate cancer progression

doi: 10.1136/jitc-2025-014276

Figure Lengend Snippet: M1-like macrophages secrete CXCL16 and support CXCR6 + CD8 + T-cell recruitment but are progressively lost during PCa progression. ( A ) UMAP plot of tumor-infiltrating myeloid cells from PCa tissues, identifying five distinct clusters, including an IL1B + macrophage subset. ( B ) Dot plot showing average expression and detection frequency of selected marker genes across macrophage and dendritic cell (DC) clusters. ( C ) Violin plots illustrating the expression of key pro-inflammatory ( IL1B , TLR2 , CD86 ), anti-inflammatory ( CD163 , MRC1 ), and chemokine ( CXCL16 ) genes across myeloid subsets. ( D ) AUCell-based quantification of M1 and M2 gene signatures across clusters; IL1B + macrophages exhibit the highest M1 signature score. Kruskal-Wallis test, ****p<0.0001. ( E ) CellChat network visualizing outgoing macrophage-derived signals to CD8 + T-cell subsets; IL1B + macrophages prominently interact with CXCR6 + TEff-like CD8 + T cells. ( F ) Bubble plot visualizing the results of ligand–receptor interaction analysis; CXCL16–CXCR6 axis ranks among the strongest predicted signals. ( G ) Gating strategy for the identification of murine bone marrow-derived macrophages (BMDMs) induced with M-CSF. ( H ) Flow cytometry of BMDMs polarized to M1 (IFN-γ+LPS) or M2 (IL-4) states, assessed by CD80 and CD206 expression. ( I ) Confocal images of THP-1-derived macrophages stained for CD68 after PMA induction. ( J ) Flow cytometry of THP-1-derived macrophages polarized to M1 (IFN-γ+LPS) or M2 (IL-4) states, assessed by MHC-II and CD206 expression. ( K ) Immunoblots showing higher CXCL16 levels in M1-polarized BMDMs compared with their M2 counterparts. ( L ) ELISA quantification of secreted CXCL16 in the supernatants of M1-polarized and M2-polarized THP-1-derived macrophages. Mann-Whitney U test, **p<0.01. ( M ) Immunoblot analysis demonstrating elevated levels of CXCL16 in M1-polarized THP-1-derived macrophages compared with M2-polarized cells. (N, O). A total of 5×10⁶ TRAMP-C1 cells suspended in 100 µL PBS were subcutaneously implanted into the right flank of 5–6-week-old male WT C57BL/6J mice (n=5 per group). Tumors were harvested at day 35 (early stage) and day 49 (advanced stage) post-inoculation. Flow cytometric analysis of TAMs revealed a significant reduction in the ratio of MHCII + CD206⁻ (M1-like) to MHCII⁻ CD206 + (M2-like) macrophages during tumor progression. Mann-Whitney U test, **p<0.01. ( P ) Multiplex immunohistochemistry of human PCa tissues (GS=3+4 vs GS=5+5) demonstrated spatial proximity between CXCL16 + M1-like macrophages (HLA-DRA + ) and CXCR6 + CD8 + T cells in lower-grade (GS=3+4) tumors, which was largely diminished in high-grade (GS=5+5) lesions. Black arrows indicate matched regions across serial tissue sections. Scale bars: upper panels, 100 µm; lower panels, 40 µm. AUCell, area under the recovery curve; GS, Gleason Score; M-CSF, macrophage colony-stimulating factor; PCa, prostate cancer; PBS, phosphate-buffered saline; TAMs, tumor-associated macrophages.

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against CD8 (Abcam, ab17147; 1:200), CXCR6 (Abcam, ab8023; 1:100), CXCL16 (ProteinTech Group, 60123-1-Ig; 1:100), CD68 (Abcam, ab125212; 1:200), iNOS (Abcam, ab3523; 1:200), HLA-DRA (ProteinTech Group, 17221-1-AP; 1:100), CD163 (Abcam, ab182422; 1:200), KLF2 (ABclonal, A16480; 1:200), FOXO1 (ProteinTech Group, 18592-1-AP; 1:100), Granzyme B (Thermo Fisher, MA1-80734; 1:100), α-SMA (Abcam, ab5694; 1:100) and AR (Abcam, ab133273; 1:100).

Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Multiplex Assay, Immunohistochemistry, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Dual regulation of CXCR6+CD8+ T cells modulates cytotoxic and exhaustion-associated programs during prostate cancer progression

doi: 10.1136/jitc-2025-014276

Figure Lengend Snippet: IL-10–STAT3–FOXO1 signaling reprograms CXCR6 + CD8 + T cells toward a dysfunctional state. ( A ) Dot plot showing the expression of IL10RA , STAT3 , STAT4 , CXCR6 , and related markers across CD8 + T cell subsets in scRNA-seq data. ( B ) IL-10 pathway activity scores across CD8 + T cell clusters. Kruskal-Wallis test, ****p<0.0001. (C, D) Murine splenic CD8 + T cells cultured in a medium containing anti-CD3 (5 µg/mL), anti-CD28 (5 µg/mL), and IL-2 (10 ng/mL) for 48 hours. Following initial activation, cells were maintained in fresh medium supplemented with IL-2 (10 ng/mL) for an additional 7 days. On day 9, cells were treated with 20 ng/mL murine IL-10, IL-15, or STAT3 inhibitor Stattic (2 µM) for 24 hours. The protein expression of STAT3, p-STATS, FOXO1, KLF2, and CXCR6 was assessed by Western blot analysis. (E, F) Flow cytometry of mouse spleen-derived CD8 + T cells shows preferential expression of IL-10R on CXCR6 + CD8 + T cells, with upregulation observed following TCR stimulation (anti-CD3/CD28+IL-2, day 10), indicating heightened susceptibility to IL-10-mediated signaling. (G–I) Flow cytometry of human peripheral blood mononuclear cell (PBMC) CD8 + T cells from healthy donors similarly demonstrates enhanced IL-10R expression on CXCR6 + CD8 + T cells and its induction on TCR stimulation. ( J ) PCA of bulk RNA-seq. Prostate tissues from Pb-Cre; Pten flox/flox ( T ) and WT mice (n=3/group) were profiled by bulk RNA-seq. PCA separated T (blue) from WT (red) chiefly along PC1 (87.24% variance) and PC2 (5.64%). ( K ) Sample-to-sample distance heatmap. Distance matrix based on transformed expression values shows tight clustering of biological replicates within genotype and clear segregation between T and WT. ( L ) Volcano plot. Differential expression analysis between T and WT (cut-offs |log2FC|≥1.5, FDR<0.05). Points are colored by direction (Up=red; Down=blue). Dashed lines indicate thresholds. Il10 , Mrc1 , Cd163 , and Cxcr6 are highlighted in purple; other selected genes are labeled as indicated. Y-axis shows –log 10 (adjusted p). ( M ) Multiplex immunofluorescence (human prostate). Representative fields from human prostate specimens (n=9). Channels: DAPI (nuclei), CD68 (pan-macrophage), HLA-DRA (M1-like macrophage), CD163 (M2-like macrophage), and IL-10. IL-10 signal is enriched in tumor regions and co-localizes with CD68 + CD163 + macrophages. Scale bar: 20 µm. DAPI, 4′,6-diamidino-2-phenylindole; FDR, false discovery rate; PCA, principal component analysis; scRNA-seq, single-cell RNA sequencing; WT, wild-type.

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against CD8 (Abcam, ab17147; 1:200), CXCR6 (Abcam, ab8023; 1:100), CXCL16 (ProteinTech Group, 60123-1-Ig; 1:100), CD68 (Abcam, ab125212; 1:200), iNOS (Abcam, ab3523; 1:200), HLA-DRA (ProteinTech Group, 17221-1-AP; 1:100), CD163 (Abcam, ab182422; 1:200), KLF2 (ABclonal, A16480; 1:200), FOXO1 (ProteinTech Group, 18592-1-AP; 1:100), Granzyme B (Thermo Fisher, MA1-80734; 1:100), α-SMA (Abcam, ab5694; 1:100) and AR (Abcam, ab133273; 1:100).

Techniques: Expressing, Activity Assay, Cell Culture, Activation Assay, Western Blot, Flow Cytometry, Derivative Assay, RNA Sequencing, Transformation Assay, Quantitative Proteomics, Labeling, Multiplex Assay, Immunofluorescence, Single Cell

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Identification of regenerating factor as a regulator of therapeutic genes for Parkinson's disease therapy. A) Conceptual diagram outlining the basis of an epigenetic regulator. B) Comparative gene expression heatmap of substantia nigra (SN) in wild type control versus 6‐OHDA‐induced Parkinson's disease (PD) mouse model. C) Heatmap showing gene expression profiles in the caudate and putamen regions of healthy individuals (HI) and a cohort of human PD patients. BG: Basal Ganglia. D) Immunofluorescence images showing TUJ1‐ and MAP2‐positive cells under each condition. Scale bar = 50 µm. E) Immunochemistry and Sholl analysis of TH‐labeled neurons. Left panel: morphology of individual neurons. Right panel: Sholl analysis showing the number of neurite intersections as a function of distance from the soma. Scale bar = 100 µm. The data are presented as mean ± SEM ( n = 5 – 6 cells per group). F) Representative traces of action potentials evoked by depolarizing current injections under each condition (sham, 6‐OHDA, 6‐OHDA+NDST3). G) Dot plot showing the top 14 GO Biological Process terms from enrichment analyses: 6‐OHDA versus Sham (left side) and 6‐OHDA+NDST3 versus 6‐OHDA (right side). H) Pearson correlation matrix of transcriptomic among samples.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Gene Expression, Control, Immunofluorescence, Labeling

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Therapeutic efficacy of NDST3 and retrograde tracing with CTB in mice. A) Schematic diagram of in vivo experimental design involving CTB injection in the PD mouse model. B) Representative immunofluorescence images of CTB, TH, and NDST3 expression in the SN of Sham, 6‐OHDA‐induced PD mice, and NDST3‐treated PD mice. Scale bar = 50 µm and 10 µm (Magnified image). C) Quantification of CTB‐, TH‐, and NDST3‐positive cells shown in Figure . Data are presented as mean ± SEM ( n = 6 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns = not significant. D) Immunofluorescence images showing GIRK2‐ and TH‐positive cells in the Sham, 6‐OHDA‐induced PD mice, and NDST3‐treated PD mice. Scale bar = 50 µm and 10 µm (Magnified image). E) 3D Z‐stack analysis (IMARIS) of TH‐positive neurons obtained via confocal microscopy. F) DAB‐DAT staining in the SN.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Drug discovery, Retrograde Tracing, In Vivo, Injection, Immunofluorescence, Expressing, Confocal Microscopy, Staining

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Efficacy and electrophysiological properties of NDST3 in chemical‐induced PD model. A) Representative traces of spontaneous firing currents recorded from DA neurons of the SNpc in brain slices from each group. B) Cumulative fractions curves showing shortened inter‐event intervals, indicating a higher frequency of spontaneous firing in the 6‐OHDA + NDST3 group compared to the 6‐OHDA group. The inner bar graph showed mean inter‐event intervals in the ipsilateral of SNpc of each group. Data are presented as mean ± SEM ( n = 6 – 8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. *** p < 0.001. C) Quantification of DA neuronal firing rates in the ipsilateral SNpc of each group. The data are presented as mean ± SEM ( n = 6–8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. * p < 0.05, and ** p < 0.01. D) Representative in vivo recording traces from the SNpc of live animals in each condition. E) Instantaneous firing frequencies during the recorded period. ( n = 4–6 independent animals per group; repeated measures) Two‐way ANOVA with Tukey's multiple comparisons test, * p < 0.05. F) Comparison of action potential waveforms among DA neurons across conditions. G) Representative image of DAB‐TH staining in ST and SN. Scale bar = 1 mm. H) Immunofluorescence images showing GIRK2‐ and TH‐positive cells in the Sham, MPTP‐induced PD mice, NDST3‐treated PD mice, and NDST3 only‐treated mice. Scale bar = 50 µm and 10 µm (Magnified image). I) Error count during the challenging beam traversal test for each experimental condition. The data are presented as mean ± SEM. ( n = 7 – 8 independent animals per group) Two‐way ANOVA with Tukey's multiple comparisons test. **** p < 0.0001. J) Errors per step during the challenging beam traversal test across conditions. The data are presented as mean ± SEM ( n = 7 – 8 independent animal per group). One‐way ANOVA with Tukey's multiple comparisons test. **** p < 0.0001. K) Fall latency in the wire‐hanging test. The data are presented as mean ± SEM ( n = 7–8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. *** p < 0.001 and **** p < 0.0001. L) Time to orient downward (T‐turn) and M) time to descend to the base (T‐total). The data are presented as mean ± SEM ( n = 7–8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: In Vivo, Comparison, Staining, Immunofluorescence

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Molecular mechanisms of NDST3 in the PD model. A) One‐way hierarchical clustering heatmap based on Z‐score of normalized expression value for 5629 genes selected with fold change ≥ 2 and raw p ‐value < 0.05. B) Principal component analysis (PCA) analysis of RNA‐seq data to visualize sample‐to‐sample variation. C) Volcano plot showing differentially expressed genes between 6‐OHDA and Sham group; Down‐regulated genes marked in blue. D) Volcano plot showing differentially expressed genes between 6‐OHDA+NDST3 and 6‐OHDA; Up‐regulated genes marked in red. E) Dot plot of top 14 GO cellular component terms from GO enrichment analyses: 6‐OHDA+NDST3 versus 6‐OHDA. Heatmap showing gene expression patterns in F) pre‐synaptic neurons, G) post‐synaptic neurons, and H) glia compartments. I) UMAP visualizing cluster identity. J) UMAP representation comparing cellular composition in 6‐OHDA and 6‐OHDA+NDST3. K) Branched trajectory analysis illustrating cell state transitions in a 2D state‐space, where each dot represents a single cell, color‐coded by group identity.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Expressing, RNA Sequencing, Gene Expression, Single Cell

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Comprehensive analysis of spatial transcriptomics and epigenetic modulation following NDST3 treatment in a PD model. A) Heatmap showing gene expression patterns in each cluster. ** p < 0.01, and **** p < 0.0001. B) Gene concept network plot displaying genes enriched in catabolic, metabolic, and wound healing GO categories. The top 30 most differentially expressed genes comparing 6‐OHDA versus Sham and 6‐OHDA+NDST3 versus 6‐OHDA. Node color intensity represents the log2 fold‐change of gene expression. C) Cell‐cell communication network plot illustrating interactions among three distinct cell clusters in 6‐OHDA‐induced PD model (left panel) and NDST3‐treated PD model (right panel), based on ligand–receptor pair probabilities using the CellChat database. Line thickness indicates proportionality to the number of interactions. D) Spatial localization of dopamine‐related markers. E) Spatial mapping of dopaminergic lineage markers identified via scRNA‐Seq. F) Heatmap visualization of CUT&RUN and ATAC‐Seq signal intensity ±2 kb around the TSS. G) Immunofluorescence images showing H3K27ac and TH‐positive cells in the Sham, 6‐OHDA‐induced PD mice, and NDST3‐treated PD mice. Scale bar = 50 µm. H) Venn diagram illustrating overlapping genes among DEGs from RNA‐Seq, scRNA‐Seq Cluster 9, CUT&RUN peak, and ATAC‐Seq peak. Average signal plot of I) CUT&RUN and J) ATAC‐seq signals at over‐enriched TSS regions of the Ncoa7 gene. K) Structure of NDST3‐NCOA7‐H3K27ac complex. Blue – NDST3, Green – NCOA7, and Red – H3K27ac. The yellow boundary represents the interaction region.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Spatial Transcriptomics, Gene Expression, Immunofluorescence, RNA Sequencing