recombinant human cxcl3 protein  (R&D Systems)

Journal: Microbiome

Article Title: Periodontitis salivary microbiota exacerbates colitis by CXCL3 derived from gut microbiota-induced macrophages

doi: 10.1186/s40168-025-02218-3

Figure Lengend Snippet: Macrophage-derived CXCL3 exacerbated colitis by macrophages M2b-like polarization in colitis mice. A The proliferation of RAW264.7 treated with CXCL3 using CCK-8 assay. B The proliferation of RAW264.7 treated with fecal supernatant and CXCL3-neutralizing antibodies using CCK-8 assay (PBS-GM, the fecal was from PBS-gavage mice; HSM-GM, the fecal was collected from HSM-gavage mice; PSM-GM, the fecal was collected from PSM-gavage mice). C Representative FACS plots of expression of CD86 and CD206 in Raw264.7 cells treated with LPS, IL4, and CXCL3. D The heatmap expression of differentially expressed genes between control macrophage (Ctrl group) and CXCL3-treated macrophage (CXCL3 group). E Volcano plot indicated differential genes expression with high fold change ( n = 3). F Functional enrichment of differential genes analyzed by KEGG in the CXCL3-treated macrophages. G and H The functions of upregulated ( G ) and downregulated ( H ) differential genes analyzed by KEGG in the CXCL3-treated macrophages. I The heatmap expression of M2-related genes in the CXCL3-treated macrophages. J Schematic representation and study design of CXCL3-treated macrophage and ABX treatment (DSS + Mø, tail vein injection of blank virus-treated macrophages in DSS mice; DSS + CXCL3-Mø, tail vein injection of CXCL3-treated macrophages in DSS mice; DSS + Mø + ABX, DSS + Mø group treated with antibiotic in DSS mice; DSS + CXCL3-Mø + ABX, DSS + CXCL3-Mø group treated with antibiotic in DSS mice). K – P The changes of the colitis level treated with CXCL3-treated macrophage and ABX, including weight loss ( K ), DAI score ( L ), representative physical and statistical diagrams of the length of the colon ( M ), histological score ( N ), representative image of hematoxylin and eosin staining ( O ), and PAS staining ( P ) ( n = 6). p values were determined by one-way ANOVA with Tukey’s multiple comparisons test ( A , B , K , and L ) and two-way ANOVA ( C , M , and N ). * p < 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, scale bar = 100 µm

Article Snippet: For PUE treatment, cells were added at a concentration of 20 μM PUE per well for 24 h. For macrophage polarization, M1 macrophages were induced by 1 μg/mL LPS, M2 macrophages were induced by 20 ng/mL IL-4 (CAT#HY-P701093, MedChemExpress), and CXCL3-treated macrophages were stimulated by 10 ng/mL CXCL3 recombinant protein.

Techniques: Derivative Assay, CCK-8 Assay, Expressing, Control, Functional Assay, Injection, Virus, Staining

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Tumor neutrophil ferroptosis was related to a distinctive subset of CD4 T cells enriched in chemoresistant tumors. A, Uniform Manifold Approximation and Projection (UMAP) visualization of tumor-infiltrating CD4 T cells from five chemosensitive and five chemoresistant breast cancers by scRNA-seq. B, Three-dimensional UMAP plot of CD4 T cells colored by chemosensitivity. C, The proportions of different subpopulations of CD4 T cells in sensitive and resistant tumors. D, Heatmap displaying scaled expression of discriminating genes for each cluster of CD4 T cells in scRNA-seq data. E, Heatmap for IL1B expression in CD4 TILs from nine chemosensitive and nine chemoresistant patients by bulk RNA-seq. F, Representative flow cytometry for IL1β and CXCL3 expression in chemosensitive and chemoresistant tumor-infiltrating CD4 T cells. G, Cell death ratio of peripheral neutrophils pretreated with the inhibitors for apoptosis, necrosis, or ferroptosis and cocultured with IL1β + CXCL3 + -d or IL1β + CXCL3 + CD4 T cells. H, Representative flow cytometric images for cellular lipid peroxidation of peripheral neutrophils cocultured with different CD4 T cells. I, Proliferation of tumor-specific CTLs upon exposure to peripheral neutrophils pretreated with IL1β + CXCL3 + or IL1β + CXCL3 + -d CD4 T cells. J, Representative immunofluorescence images (left) and quantification (right) for IL1β and CXCL3 expression in CD4 T cells in chemosensitive and chemoresistant breast cancer sections. Arrows, IL1β + CXCL3 + CD4 T cells. Scale bar, 50 μm. K, Correlation between IL1β + CXCL3 + CD4 T cells and ferroptotic neutrophils in breast tumor specimens. Results are represented as mean ± SD of n = 5 ( A–D and G–I ), n = 9 ( E and F ), or n = 468 ( J ) different patients; for K , n = 468 different patients; ***, P < 0.001 by Student t test ( F ), two-sided one-way ANOVA with the Tukey test ( G , I , and J ) or two-tailed Pearson correlation coefficient test ( K ). Quantification is shown in Supplementary Figs. S4 and S5 ( F and H ).

Article Snippet: Human IL-1β ELISA Kit (Invitrogen, #BMS224-2), Human CXCL3 ELISA Kit (Biorbyt, #orb405701), Human IL-8 ELISA Kit (R&D Systems, #D8000C), Human S100A9 ELISA kit (R&D Systems, #DY5578), Human PGE2 ELISA kit (Invitrogen, #KHL1701), Human IDO ELISA Kit (R&D Systems, #DY6030B), Human HGF ELISA Kit (R&D Systems, #DHG00B), Human TGF-beta1 ELISA Kit (R&D Systems, #DB100C), Human IL-10 ELISA Kit (R&D Systems, #D1000B), Human IL-21 ELISA Kit (R&D Systems, #DY8879), Human EBI3 ELISA Kit (R&D Systems, #DY6456), Human IL-6 ELISA Kit (R&D Systems, #D6050B), Human IL-23 ELISA Kit (R&D Systems, #D2300B), and Human IL-17 ELISA Kit (R&D Systems, #QK317) were utilized following the manufacturer’s instructions.

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Immunofluorescence, Two Tailed Test

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Cross-talk between neutrophils and Fer-CD4 T cells maintained extensive neutrophil ferroptosis. A, Gene Ontology (GO) terms associated with upregulated genes of C2_IL1B cluster in scRNA-seq. B, Gene set enrichment analysis of bulk RNA-seq revealed enrichment of neutrophil chemotaxis genes in chemoresistant CD4 TILs. C, Scheme of chemotaxis assays with Boyden transwell chambers. D, Representative images (left) and quantification (right) of chemotaxis assays for peripheral neutrophils toward CM of different CD4 TILs. Scale bar, 100 μm. E, Migration tracks of neutrophils in μ-slide chemotaxis experiments toward CM of non–Fer-CD4 or Fer-CD4 T cells. F, Representative fluorescent images of cytoskeleton staining (left) and quantification (right) of filopodium-like protrusions (FLP) of peripheral neutrophils in the presence of CM from non–Fer-CD4 or Fer-CD4 T cells. Scale bar, 5 μm. G, Ex vivo tumor slice migration assays for the recruitment of CFSE-labeled neutrophils into breast tumor slices with high or low density of Fer-CD4 T cells. Scale bar, 100 μm. H, Dot plot for the expression of various chemokines in C2_IL1B cells in scRNA-seq. I, ELISA for CXCL3, IL8, and S100A9 release of non–Fer-CD4 or Fer-CD4 T cells. J and K, Migration ( J ) and filopodium-like protrusions ( K ) of peripheral neutrophils in the presence of CM from Fer-CD4 T cells and different antibodies. L, Cell death of peripheral neutrophils in the presence of CM of Fer-CD4 T cells and different antibodies categorized by location (top or bottom chamber of transwell system). M, CXCL3 and IL1β protein level of naïve CD4 T cells by immunoblotting after coculturing with control or ferroptotic neutrophils, which were generated by engagement with non–Fer-CD4 or Fer-CD4 T cells, respectively. Results are represented as mean ± SD of n = 7 ( D ) or n = 5 ( F and I–M ) or n = 6 ( G ). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test ( D , F , and J–L ) or Student t test ( G , I , and M ). Representative images are shown in Supplementary Fig. S7 ( J and K ).

Article Snippet: Human IL-1β ELISA Kit (Invitrogen, #BMS224-2), Human CXCL3 ELISA Kit (Biorbyt, #orb405701), Human IL-8 ELISA Kit (R&D Systems, #D8000C), Human S100A9 ELISA kit (R&D Systems, #DY5578), Human PGE2 ELISA kit (Invitrogen, #KHL1701), Human IDO ELISA Kit (R&D Systems, #DY6030B), Human HGF ELISA Kit (R&D Systems, #DHG00B), Human TGF-beta1 ELISA Kit (R&D Systems, #DB100C), Human IL-10 ELISA Kit (R&D Systems, #D1000B), Human IL-21 ELISA Kit (R&D Systems, #DY8879), Human EBI3 ELISA Kit (R&D Systems, #DY6456), Human IL-6 ELISA Kit (R&D Systems, #D6050B), Human IL-23 ELISA Kit (R&D Systems, #D2300B), and Human IL-17 ELISA Kit (R&D Systems, #QK317) were utilized following the manufacturer’s instructions.

Techniques: RNA Sequencing, Chemotaxis Assay, Migration, Staining, Ex Vivo, Labeling, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Generated

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Tumor neutrophil ferroptosis was related to a distinctive subset of CD4 T cells enriched in chemoresistant tumors. A, Uniform Manifold Approximation and Projection (UMAP) visualization of tumor-infiltrating CD4 T cells from five chemosensitive and five chemoresistant breast cancers by scRNA-seq. B, Three-dimensional UMAP plot of CD4 T cells colored by chemosensitivity. C, The proportions of different subpopulations of CD4 T cells in sensitive and resistant tumors. D, Heatmap displaying scaled expression of discriminating genes for each cluster of CD4 T cells in scRNA-seq data. E, Heatmap for IL1B expression in CD4 TILs from nine chemosensitive and nine chemoresistant patients by bulk RNA-seq. F, Representative flow cytometry for IL1β and CXCL3 expression in chemosensitive and chemoresistant tumor-infiltrating CD4 T cells. G, Cell death ratio of peripheral neutrophils pretreated with the inhibitors for apoptosis, necrosis, or ferroptosis and cocultured with IL1β + CXCL3 + -d or IL1β + CXCL3 + CD4 T cells. H, Representative flow cytometric images for cellular lipid peroxidation of peripheral neutrophils cocultured with different CD4 T cells. I, Proliferation of tumor-specific CTLs upon exposure to peripheral neutrophils pretreated with IL1β + CXCL3 + or IL1β + CXCL3 + -d CD4 T cells. J, Representative immunofluorescence images (left) and quantification (right) for IL1β and CXCL3 expression in CD4 T cells in chemosensitive and chemoresistant breast cancer sections. Arrows, IL1β + CXCL3 + CD4 T cells. Scale bar, 50 μm. K, Correlation between IL1β + CXCL3 + CD4 T cells and ferroptotic neutrophils in breast tumor specimens. Results are represented as mean ± SD of n = 5 ( A–D and G–I ), n = 9 ( E and F ), or n = 468 ( J ) different patients; for K , n = 468 different patients; ***, P < 0.001 by Student t test ( F ), two-sided one-way ANOVA with the Tukey test ( G , I , and J ) or two-tailed Pearson correlation coefficient test ( K ). Quantification is shown in Supplementary Figs. S4 and S5 ( F and H ).

Article Snippet: The primary antibodies used included rabbit anti-human CD4 (1:100, Abcam, cat. #ab133616, RRID: AB_2750883), rabbit anti-human CXCL3 (1:200, Biorbyt, cat. #orb13448, RRID: AB_10748863), rabbit anti-mouse CXCL3 (1:100, Abcam, cat. #ab220431, RRID: AB_2938758), rabbit anti-human/mouse IL1β (1:50, Abcam, cat. #ab254360, RRID: AB_2936299), goat anti-human/mouse MPO (10 μg/mL, R&D Systems, cat. #AF3667, RRID: AB_2250866), mouse anti-4 HNE (1:25, Abcam, cat. #ab48506, RRID: AB_867452), rabbit anti-human IL1R1 (1:100, Abcam, cat. #ab106278, RRID: AB_10865509), rabbit anti-human/mouse p65 (1:500, Cell Signaling Technology, cat. #8242S, RRID: AB_10859369), mouse anti-human/mouse cytokeratin (1 μg/mL, Abcam, cat. #ab7753, RRID: AB_306047), rabbit anti-mouse CD8 (1:100, Abcam, #ab203035), and rabbit anti-human/mouse granzyme B (1:200, Abcam, cat. #ab255598, RRID: AB_2860567).

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Immunofluorescence, Two Tailed Test

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Cross-talk between neutrophils and Fer-CD4 T cells maintained extensive neutrophil ferroptosis. A, Gene Ontology (GO) terms associated with upregulated genes of C2_IL1B cluster in scRNA-seq. B, Gene set enrichment analysis of bulk RNA-seq revealed enrichment of neutrophil chemotaxis genes in chemoresistant CD4 TILs. C, Scheme of chemotaxis assays with Boyden transwell chambers. D, Representative images (left) and quantification (right) of chemotaxis assays for peripheral neutrophils toward CM of different CD4 TILs. Scale bar, 100 μm. E, Migration tracks of neutrophils in μ-slide chemotaxis experiments toward CM of non–Fer-CD4 or Fer-CD4 T cells. F, Representative fluorescent images of cytoskeleton staining (left) and quantification (right) of filopodium-like protrusions (FLP) of peripheral neutrophils in the presence of CM from non–Fer-CD4 or Fer-CD4 T cells. Scale bar, 5 μm. G, Ex vivo tumor slice migration assays for the recruitment of CFSE-labeled neutrophils into breast tumor slices with high or low density of Fer-CD4 T cells. Scale bar, 100 μm. H, Dot plot for the expression of various chemokines in C2_IL1B cells in scRNA-seq. I, ELISA for CXCL3, IL8, and S100A9 release of non–Fer-CD4 or Fer-CD4 T cells. J and K, Migration ( J ) and filopodium-like protrusions ( K ) of peripheral neutrophils in the presence of CM from Fer-CD4 T cells and different antibodies. L, Cell death of peripheral neutrophils in the presence of CM of Fer-CD4 T cells and different antibodies categorized by location (top or bottom chamber of transwell system). M, CXCL3 and IL1β protein level of naïve CD4 T cells by immunoblotting after coculturing with control or ferroptotic neutrophils, which were generated by engagement with non–Fer-CD4 or Fer-CD4 T cells, respectively. Results are represented as mean ± SD of n = 7 ( D ) or n = 5 ( F and I–M ) or n = 6 ( G ). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test ( D , F , and J–L ) or Student t test ( G , I , and M ). Representative images are shown in Supplementary Fig. S7 ( J and K ).

Article Snippet: The primary antibodies used included rabbit anti-human CD4 (1:100, Abcam, cat. #ab133616, RRID: AB_2750883), rabbit anti-human CXCL3 (1:200, Biorbyt, cat. #orb13448, RRID: AB_10748863), rabbit anti-mouse CXCL3 (1:100, Abcam, cat. #ab220431, RRID: AB_2938758), rabbit anti-human/mouse IL1β (1:50, Abcam, cat. #ab254360, RRID: AB_2936299), goat anti-human/mouse MPO (10 μg/mL, R&D Systems, cat. #AF3667, RRID: AB_2250866), mouse anti-4 HNE (1:25, Abcam, cat. #ab48506, RRID: AB_867452), rabbit anti-human IL1R1 (1:100, Abcam, cat. #ab106278, RRID: AB_10865509), rabbit anti-human/mouse p65 (1:500, Cell Signaling Technology, cat. #8242S, RRID: AB_10859369), mouse anti-human/mouse cytokeratin (1 μg/mL, Abcam, cat. #ab7753, RRID: AB_306047), rabbit anti-mouse CD8 (1:100, Abcam, #ab203035), and rabbit anti-human/mouse granzyme B (1:200, Abcam, cat. #ab255598, RRID: AB_2860567).

Techniques: RNA Sequencing, Chemotaxis Assay, Migration, Staining, Ex Vivo, Labeling, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Generated