Journal: eLife

Article Title: A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx

doi: 10.7554/eLife.72849

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse CXCL3 ELISA Kit , Rockland , Cat#:KOA0825 , .

Techniques: Flow Cytometry, cDNA Library Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Staining, Blocking Assay, In Vivo, Control

Journal: PLoS ONE

Article Title: Cancer cell-derived interleukin-33 decoy receptor sST2 enhances orthotopic tumor growth in a murine pancreatic cancer model

doi: 10.1371/journal.pone.0232230

Figure Lengend Snippet: (A) The amount of CXCL3 in Panc02-shCont (n = 9) and Panc02-sh#5 (n = 9) tumor tissues. (B) IHC analysis of MPO + neutrophil infiltration in Panc02-shCont and Panc02-sh#5 tumors. Bar: 50 μm. (C) The number of MPO + cells per field (×400 magnification) in Panc02-shCont (n = 9 fields of 2 tissue sections) and Panc02-sh#5 tumors (n = 12 fields of 2 tissue sections). (D) RT-qPCR analysis of expression of the neutrophil marker gene Ly6G in Panc02-shCont (n = 5 tumors) and Panc02-sh#5 tumors (n = 6 tumors). (E) The number of F4/80 + cells per field (×400 magnification) in Panc02-shCont (n = 10 fields of 5 tissue sections) and Panc02-sh#5 tumors (n = 12 fields of 4 tissue sections). (F) The number of CD20 + cells per field (×400 magnification) in Panc02-shCont (n = 9 fields of 3 tissue sections) and Panc02-sh#5 tumors (n = 10 fields of 3 tissue sections). (G, H) Angiogenesis in Panc02-shCont and Panc02-sh#5 tumors. G: CD31 staining. Bar: 50 μm. H: Vessel density. Panc02-shCont (n = 11 fields) and Panc02-sh#5 cells (n = 14 fields). The data are shown as the mean ± SD. *P<0.05. ns, not significant.

Article Snippet: Mouse CXCL3 concentrations were measured with the Cxcl3 ELISA Kit (Mouse) (Aviva Systems Biology, Corp. San Diego, CA, USA) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Expressing, Marker, Staining

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: (a ) Western blotting analysis of CD133, secreted and endogenous CXCL3 proteins expression in HCC cell lines. The gels in the same panel mentioned in this article had been run under the same experimental conditions. Uncropped full-length blots were showed in the . ( b ) CD133 + HCC cells expressed higher level of secreted and endogenous CXCL3 proteins compared with the corresponding CD133 − cells. Uncropped full-length blots were showed in the .

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) MTT assays showed that down-regulation of CXCL3 expression by shRNA inhibited cell growth in Hep3B, Huh7 and PLC/PRF/5 cells after day 4 (values were represented as the mean ± SD; * p < 0.05 vs shNC control, the Bonferroni method was used for the multiple comparison). ( b ) Down-regulation of CXCL3 inhibited cell growth in HCC-LY5, SMMC-7721 and MHCC-LM3 cells after day 4 (values were represented as the mean ± SD; * p < 0.05 vs shNC control, the Bonferroni method was used for the multiple comparison). ( c ) Results of the clone-formation assay in CXCL3 knockdown CD133 + /CD133 − HCC cells sorted from HCC-LY5 ( p = 0.0005) and SMMC-7721 ( p = 0.028) (values were represented as the mean ± SD; * p < 0.05; t -test, vs cells treated with shNC). ( d ) Tumor-sphere formation analysis displayed that CXCL3-silence decreased the tumor-sphere number in Huh7 ( p = 0.0325) and PLC/PRF/5 ( p = 0.0491) CD133 + cells (values were represented as the mean ± SD; * p < 0.05; t -test, vs cells treated with shNC). ( e ) The weight of tumors from BALB/c (nu/nu) mice injected with CXCL3 knockdown or control SMMC-7721 cells are shown (CD133 + -NC vs CD133 − -NC, p = 0.045; CD133 + -NC vs CD133 + -shCXCL3, p = 0.037) (values were represented as the mean ± SD; * p < 0.05 vs cells treated with shNC, the Bonferroni method was used for the multiple comparison).

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Expressing, shRNA, Control, Comparison, Tube Formation Assay, Knockdown, Injection

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) 100 ng/ml exogenous CXCL3 treatment induced Erk1/2 phosphorylation in a time dependent manner in Huh7, HCC-LY5 and SMMC-7721 cells. Uncropped full-length blots were showed in the . ( b ) Long time treatment of CXCL3 (100 ng/ml) induced Erk1/2 and EST1 phosphorylation, and promoted CD133 expression in HCC cells. Uncropped full-length blots were showed in the .

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Phospho-proteomics, Expressing

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) CD133 was overexpressed in HCC-LY5 and SMMC-7721 cells and the luciferase activities associated with CXCL3 promoter are shown. Reporter gene activities are expressed as fold changes relative to the control ( p = 0.002 in HCC-LY5; p = 0.003 in SMMC-7721) (values were represented as the mean ± SD; * p < 0.05; t -test, vs vector control). ( b ) CD133 overexpression up-regulated CXCL3 protein expression in HCC cell lines. Uncropped full-length blots were showed in the . ( c ) CD133 knockdown down-regulated CXCL3 expression in HCC cells. Uncropped full-length blots were showed in the .

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Luciferase, Control, Plasmid Preparation, Over Expression, Expressing, Knockdown

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) Realtime PCR analysis demonstrated that HCC tumor tissue samples expressed higher level of CXCL3 mRNA (22/30) compared with the para-tumor tissue samples. ( b ) Western blotting analysis of 7 pair tissue samples showed that CXCL3 HCC tumor tissue samples (T) expressed higher level of CXCL3 protein compared with the para-tumor tissue samples (N). Uncropped full-length blots were showed in the . ( c ) ELISA results displayed that serum CXCL3 lever was higher in HCC patients ( p = 0.0418)(values were represented as the mean ± SD; ** p < 0.01; t -test, vs normal people control). ( d ) The cutoff value of 66.36 pg/ml for serum CXCL3 with a sensitivity of 62.4% and specificity of 88.1% using receiver operating characteristic analysis. ( e ) Results of overall survival analysis of serum CXCL3 in HCC patients are shown (Kaplan-Meier analysis).

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: Correlation between Clinicopathologic Features and Serum CXCL3 Protein Expression.

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: Immune Antibodies and Helminth Products Drive CXCR2-Dependent Macrophage-Myofibroblast Crosstalk to Promote Intestinal Repair

doi: 10.1371/journal.ppat.1004778

Figure Lengend Snippet: Bone marrow derived MΦ (BMM) or primary small intestinal MF from were co-cultured with Hpb larvae (L3) for 24h with our without immune sera (IS) from challenge Hpb infected mice; (A) CXCL2 in BMM culture supernatants (C57BL/6 or Fcrg -/- ) cultured without (ctr) or with IS, L3 or both; (B) top: CXCL2 staining in control or L3IS-co-cultured BMM, bottom: IgG (red)/ CXCL2 (green) overlay; (C) Scratch wound closure by MF after addition of supernatants from BMM, cultured without (ctr) or with L3 and IS +/- CXCR2 antagonist SB265610. (D) Cxcl2 mRNA induction in MF after IS, L3 or L3 and IS-stimulation relative to unstimulated MF; (E) CXCL2 in culture supernatants from MF, cultured without (ctr) or with L3 or L3 and IS; (F) Overlays of CXCL2 (green) and IgG (red) for unstimulated (control) MF and L3IS-co-cultured C57BL/6 or Fcrg -/- MF; (G) Cxcl2 mRNA induction in MF after L3 co-culture; (H) CXCL2 in MF supernatants after culture without (ctr) or with L3 or L3IS;(I) Cxcl3 mRNA induction in MF after culture in the absence or presence of L3 or L3IS; (J) CXCL3 in unstimulated (control) MF or L3-co-cultured MF; (K) Cxcl3 mRNA induction in MF after L3-co-culture; All data are pooled from at least 2 independent experiments with cells from 3–4 mice per group and presented as mean + SEM.

Article Snippet: Human CXCL3 was quantified using an anti-human CXCL3 ELISA Kit (LuBioScience, Luzern, Switzerland).

Techniques: Derivative Assay, Cell Culture, Infection, Staining, Control, Co-Culture Assay

Journal: PLoS Pathogens

Article Title: Immune Antibodies and Helminth Products Drive CXCR2-Dependent Macrophage-Myofibroblast Crosstalk to Promote Intestinal Repair

doi: 10.1371/journal.ppat.1004778

Figure Lengend Snippet: (A) Human MDM from 3 healthy blood donors (1–3) were co-cultured with A . suum larvae in the absence or presence of IS from challenge- A . suum infected pigs and time-lapse movies were recorded (2 frames/ s, 1 min); upper panel: representative snap shots at T = 0; lower panel: representative temporal color code images (120 frames, 1 min)—colors represent different time points; Scale bars 100 μm; (B) Adherent MDM per A . suum larva were counted in time-lapse movies; (C) CXCL3 in culture supernatants from human MDM cultured with or without immune sera from Ascaris -infected pigs (IS), Ascaris suum larvae ( A . s .) or both ( A . s . IS) quantified by ELISA; (D) Scratch wound closure by human MF cultured in the presence of conditioned media from MDM (MDM sup) stimulated with A . s . IS +/- the CXCR2 antagonist SB265610; All data are pooled from at least 3 independent experiments with n = 3 co-cultures in each and presented as mean +/-SEM.

Article Snippet: Human CXCL3 was quantified using an anti-human CXCL3 ELISA Kit (LuBioScience, Luzern, Switzerland).

Techniques: Cell Culture, Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response

doi: 10.1186/s12974-016-0753-x

Figure Lengend Snippet: Cytokine expression changes at 7 days after dMCAO. a Cytokine gene profiling at 7 days after dMCAO, inhibitor versus control groups. Clustergram of 84 common cytokines grouped by sample type (i1, i2, i3—samples collected from three different mice from the inhibitor group. c1, c2, c3—three samples from control group). Non-supervised hierarchical clustering was used to display common cytokine gene expression by heat map visualization, with dendrograms indicating coregulated genes across groups or individual samples. Results for two statistically different (between inhibitor and controls) genes Ccl12 and Cxcl3 are enlarged and showed in the bottom left corner . A color bar ( bottom ) relates color code to the magnitude of the differences in gene expression relative to the all-sample means for each gene. The results are also demonstrated in the form of scatter plots ( b ) of 84 common cytokine assays, revealing four upregulated and two downregulated genes (with more than twofold difference in expression between the inhibitor and control groups). Orange symbols identify upregulated genes, and blue symbols identify downregulated genes. c Protein expression levels of CCL12 and CXCL3 cytokines were assessed in the lesioned hemispheres of the animals from control ( red plots ) and inhibitor ( blue plots ) groups at 7,14, and 21 days after dMCAO, using specialized Mouse CCL12 and CXCL3 PicoKine™ ELISA kits (Boster Biological Technology). Cytokine expressions were also measured in the lesioned hemisphere of dMCAO-subjected mice at 48 h post-dMCAO ( orange diamond ). N = 6 mice per group/per time point. Two-way ANOVA followed by Tukey’s multiple comparison test; * p < 0.05. Error bars : SEM

Article Snippet: Expression levels of CCL12 and CXCL3 were detected using Mouse CCL12/MCP5 PicoKineTM (Boster Biological Technology, Cat# EK1128) and Mouse CXCL3 PicoKineTM ELISA Kits (Boster Biological Technology, Cat# EK1364), according to manufacturer’s recommendations.

Techniques: Expressing, Control, Gene Expression, Enzyme-linked Immunosorbent Assay, Comparison

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