Journal: Mucosal Immunology

Article Title: Butyrate regulates neutrophil homeostasis and impairs early antimicrobial activity in the lung

doi: 10.1016/j.mucimm.2023.05.005

Figure Lengend Snippet: Effect of butyrate on acute Pseudomonas aeruginosa immunity. (A) Quantification of the total number of cells in BALF 18 hours post-inoculation with 1 × 10 6 CFU of P. aeruginosa strain PAO1 in Ctrl and But mice. (B) Frequency and quantification of cell types in cytospins of BALF from control and butyrate-treated mice 18 hours after PAO1 infection; (C) Quantification of P. aeruginosa CFU in BALF 18 hours post-challenge. (D, E) Cytokine and chemokine production in BALF 18 hours following infection. (F) MFI of CXCR2 on lung AM and IM from Ctrl and But mice. (G) MFI of CXCR2 on Ctrl and But lung macrophages US or stimulated for 2 hours with LPS in vitro . (H) transmigration of Ctrl and But neutrophils toward a CXCL2 or CCL2 chemokine gradient after 3 hours. Results are a mean of two independent experiments. Values are expressed as mean ± standard error of mean; n = 6–14. Statistical significance was determined with one-way analysis of variance in (A–E, G, H) and Student’s t test (unpaired, two-tailed) in (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. AM = alveolar macrophages; BALF = bronchoalveolar lavage fluid; But = butyrate-treated; CFU = colony-forming units; Ctrl = control; IFN = interferon; IL = interleukin; IM = interstitial macrophages; LPS = lipopolysaccharide; MFI = mean fluorescence intensity; PAO1 = P. aeruginosa strain 1; TNF = tumor necrosis factor; US = unstimulated.

Article Snippet: To differentiate progenitor and neutrophil populations in the BM, cells were stained with antibodies to lineage cocktail, CD115 (BD Biosciences, clone 2B8), SiglecF (BD Biosciences, clone 2B8), CD117 BUV117 (BD Biosciences, clone 2B8), Sca1 APCFire (Biolegend, clone D7), FcgR biot (Miltenyi, clone 93), CD34 BV421 (Biolegend, clone SA376A4), CD11b BV510 (BD Biosciences, clone M1/70), Ly6G Pecy5 (eBioscience, clone 1A8-Ly6g), CXCR2 Pecy7 (Miltenyi, clone REA942) and streptavidin BUV395 (BD Biosciences).

Techniques: Control, Infection, In Vitro, Transmigration Assay, Two Tailed Test, Fluorescence

Journal: Advanced Science

Article Title: Interior Decorative VOCs Elevate T Cell‐Mediated Obstructive Lung Disease Risks via Osteogenesis‐Driven Lymphoid‐Biased Hematopoiesis

doi: 10.1002/advs.202512663

Figure Lengend Snippet: VOCs exposure enhances T cell maturation and their recruitment to lung tissues. Mice were exposed to CON or VOCs for 8‐week using the whole‐body inhalation exposure model. A) Representative images of H&E‐stained thymic sections. Black dashed lines indicate the cortical thickness of thymus. Scale bar: 100 µm. Immunohistochemical analysis for B) CD3 and C) CD8 in thymic sections, and the percentage of positive area. Yellow arrows indicate the positive expression. Scale bar: 40 µm. D) Representative flow cytometric dot plots and quantitative analysis of E) CD4 + T and F) CD8 + T cells in the PB. G) Immunostaining for the colocalization of CXCR3 (red) and CD8 (purple) in lung sections, and the corresponding quantitative analysis. Yellow arrows indicate CXCR3 colocalization with CD8. Scale bar: 20 µm. H) Immunostaining for the colocalization of CCR5 (green) and CD8 (purple) in lung sections, and the corresponding quantitative analysis. Yellow arrows indicate CCR5 colocalization with CD8. Scale bar: 20 µm. I) Quantitative analysis of CXCL9, CXCL10, and CXCL11 in the PB by ELISA assay. Protein expressions of CXCL10 in J) BALF and K) LI. L) A schematic representation summarizing the T cell maturation and recruitment to lung tissue. Statistical analysis was performed using two‐tailed unpaired t ‐test (A–C and E–K). * p < 0.05 and ** p < 0.01. Data are expressed as mean ± SEM ( n ≥ 3).

Article Snippet: Similarly, mouse lung sections were incubated with primary antibodies against CXCR3 (Bosterbio, China), CCR5 (Servicebio, China), and CD8 (Servicebio, China) at 4 °C overnight, followed by the application of secondary antibodies.

Techniques: Staining, Immunohistochemical staining, Expressing, Immunostaining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: (A) Flow cytometry analysis of CXCR3 protein expression on a VGP melanoma cell line, C9. Cells were incubated with serum at 5% CO 2 . (B) Graph of CXCR3 protein expression on BOWES cells from 8 separate flow cytometry experiments. Cells were cultured either in normal culture conditions (far left bar), in serum-free media at 8% CO 2 for 24, 48, or 72 hrs, or in serum-free media at 8% CO 2 for 48 hrs followed by 48 hrs in normal culture conditions (far right bar). T-tests were performed on the averages for each time point, comparing treatment cells with the 0hr time point (NS = P>0.05, *** = P≤ 0.001). (C-D) Flow cytometry analysis of CXCR3 protein expression on an RGP cell line, BOWES. Cells were incubated (C) with serum at 5% CO 2 or (D) without serum at 8% CO 2 for 72 hrs. All flow cytometry plots and graphs are representative data from at least 3 separate experiments. Red lines = unstained cells, blue lines = cells stained with CXCR3.

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Incubation, Cell Culture, Staining

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: Gene expression of stressed BOWES cells (cultured in serum-free media at 8% CO 2 ) relative to those cultured under normal serum-containing conditions (10% FBS media at 5% CO 2 ).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Gene Expression, Cell Culture

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: (A) BOWES cells were cultured for 48 hrs under stressful conditions (serum-free media at 8% CO 2 ) in the presence of ligand (100ng/ml CXCL9 and CXCL10), with the addition of DMSO, 0.2μM, or 1μM AMG487. IL-8 expression was measured with RT-PCR, fold change was calculated relative to cells treated 1μM AMG487. (B) Flow cytometry analysis of representative data from 3 separate sorts. CXCR3 Low and CXCR3 High cells were sorted based on the gates shown. Red line = unstained cells, blue line = cells stained with CXCR3. (C) CXCR3 and (D) IL-8 RT-PCR results from unsorted cells grown with serum in 5% CO 2 or without serum in 8% CO 2 (stressed), and sorted stressed cells (CXCR3 Low and CXCR3 High ). Fold change was calculated relative to unsorted stressed cells. T-tests were performed on the relative fold changes, pooled data from 3 separate experiments (* = P≤ 0.05, ** = P≤0.01, ***P≤ 0.001).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: BOWES cells stably transfected with CXCR3 or an empty vector (PCMV6) were grown in serum-containing media. (A) Flow cytometry and (B) RT-PCR were used to measure CXCR3 expression. (A) BOWES WT (red line), BOWES PCMV6 clone #1(blue line), and BOWES CXCR3 clones #1 (orange line), #2 (light green), and #3 (dark green line). (B) RT-PCR was normalized to CXCR3 clone #2. (C) BOWES PCMV6 and CXCR3 cells were grown in serum-free media for 2 hrs, then in the presence of CXCL9 and CXCL10 for 2, 5, 10, 20, or 30 minutes. Total protein was isolated and probed for pERK and total ERK via immunoblot analysis. (D) BOWES PCMV6 and CXCR3 cells were plated on a membrane above serum-free media with CXCL9/10 (+ligand) or without (-ligand), and total migrated cells per field (40X objective) were quantified after 6hrs. Cells were also plated on the membrane in the presence or absence of AMG487. T-tests were performed on the BOWES CXCR3 migrated cells ± ligand in the presence and absence of AMG487 (NS = P>0.05, **** = P≤ 0.0001). (E) BOWES PCMV6 and CXCR3 cells were grown in serum-containing media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487. Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to BOWES PCMV6 0 μM. T-tests were performed on the relative fold changes, representative data from 3 separate experiments (**** = P≤ 0.0001).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay, Isolation, Western Blot, Membrane

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: WM35 IL-8 signaling is CXCR3-signaling independent. (A) BOWES WT cells were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487 and with DMSO (black bars) or 3 μM U0126 (gray bars). Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to cells treated with 0 μM U0126 and 1 μM AMG487. (B-C) Flow cytometry analysis of CXCR3 protein expression on the BRAF V600E RGP cell line, WM35. Cells were incubated (B) with 5% serum at 5% CO 2 or (C) without serum at 8% CO 2 for 72 hrs. Red lines = unstained cells, blue lines = cells stained with CXCR3. (D) BOWES WT (black bars) and WM35 cells (gray bars) were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487. Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to BOWES cells treated with 0 μM AMG487. (E) WM35 cells were grown in serum-free media for 48 hrs in the presence of ligand, with 0, 0.2, or 1 μM AMG487 and either DMSO (black bars), 3 μM U0126 (striped bars), or 3 μM PLX4032 (gray bars). Expression of IL-8 was measured via RT-PCR, fold change was calculated relative to WM35 cells treated with 0 μM AMG487 and 3 μM U0126. All T-tests were performed on the relative fold changes, all data is representative from at least 3 separate experiments (NS = P>0.5, * = P≤ 0.05, ** = P≤0.01, **** = P≤ 0.0001).

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Incubation, Staining

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: Tumor growth in vivo .

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: In Vivo, Injection

Journal: PLoS ONE

Article Title: CXCR3 Signaling in BRAF WT Melanoma Increases IL-8 Expression and Tumorigenicity

doi: 10.1371/journal.pone.0121140

Figure Lengend Snippet: BOWES PCMV6 and CXCR3 cells were injected interdermally into NSG mice (5 x 10 5 cells per injection). Tumors were measured weekly with calipers and tumor volume (mm 3 ) was calculated. Mean tumor volume and SEM were calculated from mice that had measurable tumors. (A) The graph shows mean tumor volume (with SEM) over the 6 week period. Linear regression analysis was performed on the two slopes (P = 0.006). (B) Tumor tissue from injected mice was resected at the time of harvest and RNA was isolated. IL-8 expression was measured in BOWES PCMV6 and CXCR3 tumor tissue via RT-PCR (**** = P≤ 0.0001). Representative individual tumors are presented in graph.

Article Snippet: Cells were transfected with a human CXCR3 ORF construct (Origene, Rockville, MD, USA) (under control of the CMV promoter, with Neomycin resistance) or a pCMV6 empty vector control (Origene), using Lipofectamine 3000 (Life Technologies), according to the manufacturer’s instructions.

Techniques: Injection, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 1. CXCR2 is a surface marker for NE cells in human PCa and is associated with disease progression. (A) Representative hematoxylin and eosin (H&E) staining (left) and IHC for NE marker CHGA (right) of human primary PCa. Nuclei (DAPI staining) are shown in blue. (B) Representative immunofluorescence images of CXCR2 (red) and NE marker CHGA (green) staining in human primary PCa. White arrowheads point to CXCR2+ CHGA+ NE tumor cells. (C) Immunofluorescence of CXCR2 (red) and luminal marker KRT8 (green) in human primary prostate adenocarcinoma. White arrowheads indicate CXCR2+ NE cells (red), which are negative for KRT8 (green). (D and E) Representative images (D) and quantification (E) of CXCR2 IHC staining on TMAs. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and interquartile range. (F) Analysis of CXCR2 expres- sion among primary and metastatic PCa tumors from the dataset of Taylor et al. (11). Logistic regression analysis was performed using t test; lines represent means ± SD.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Marker, Biomarker Discovery, Staining, Immunofluorescence, Immunohistochemistry, MANN-WHITNEY

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 2. Distinct signaling was observed between CXCR2+ NE and CXCR2− luminal cells isolated from human primary PCa. (A) Experimental scheme for gene expression and cellular function analysis of human PCa CXCR2+ NE and CXCR2− luminal populations. (B) Volcano plot of differentially expressed genes between CXCR2+ NE and CXCR2− luminal cells isolated from fresh primary human PCa tissue. (C) Heat map of genes from sorted benign basal cells (ITGA6+), NE tumor cells (CXCR2+), and luminal-type tumor cells (CXCR2−). (D and E) Tumor organoids generated by CXCR2+ NE cells (right) and CXCR2− luminal cells (left). Representative images (D) and quantification (E) of three independent patients’ samples are shown. 3D, three-dimensional. (F) Signaling pathways associated with CXCR2 expression in PCa. Gene expression of 459 primary PCa was obtained from TCGA prostate adenocarcinoma database (TCGA-PRAD). Ingenuity pathway analysis was performed using 682 genes that were correlated with CXCR2 expression (P < 0.05, Pearson’s correlation). (G and H) The enrichment scores of NE gene sets (G) and prostate adenocarcinoma gene sets (H) in CXCR2+ NE cells isolated from primary human PCa tissue (18). FDR, false discovery rate; NES, normalized enrichment score. (I) Heat map of gene expression among CXCR2+ NE and CXCR2− luminal tumor cells from fresh primary human PCa tissue, SCNC/neuroendocrine prostate cancer (NEPC), and prostate adenocarcinoma (18).

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Isolation, Gene Expression, Cell Function Assay, Generated, Protein-Protein interactions, Expressing

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 3. CXCR2-mediated phenotypic switch drives therapy resistance in PCa. (A) Flow cytometric quantification of CXCR2+ cells in parental LNCaP cell line (left) and LNCaP cells cultured in charcoal- stripped (androgen-deprived) medium for 2 weeks (right). Representative images of three independent experiments are shown. (B) Flow cytometric quantification of CXCR2+ cells in parental C4-2B cell line (left) and C4-2B cells treated with enzalutamide (20 M) for 2 weeks (right). Representative images of three independent experiments are shown. (C and D) Parental LNCaP cells and LNCaP cells overexpressing CXCR2 (LNCaP- CXCR2) were treated without (left) or with (right) enzalutamide (ENZA) for 2 weeks. Representative image (C) and quantification (D) of colony formation assay are shown. cDNA, complementary DNA. (E and F) CXCR2 expression induces a lineage switch from the luminal phenotype (AR+, KLK3+, and CHGA−) to the NE phenotype (AR−, KLK3−, and CHGA+) in an in vivo xenograft model. Representative images (E) and quantifica- tion (F) are shown. (G) Heat map of selected differentially expressed genes between LNCaP cells and LNCaP-CXCR2 cells. (H) Enrichment for binding sites for luminal-defining transcription factors in LNCaP cells measured by ChIP-seq analyses. The red dot represents the query signature of 1000 up- and down-regulated genes between LNCaP and LNCaP-CXCR2 cells. The blue dot represents one of a total of 100,000 randomly sampled gene lists of equal size to the query signature. The rank of the query gene list divided by the total number of resample instances was then used as the P value for the probability of enrichment by chance. (I) mRNA ex- pression of luminal markers in LNCaP cells with/without CXCR2 overexpression or C4-2B cells with/without CRISPR-Cas9 knock- out of CXCR2. (J) Growth curves of C4-2B/ MDVR-sgCtrl and C4-2B/MDVR-sgCXCR2 (CRISPR-Cas9 knockout of CXCR2) with or without enzalutamide (20 M) treatment. (K) The enrichment score of NE gene sets (18) in LNCaP-CXCR2 cell. (L) A model demon- strating how CXCR2 drives NE phenotype in PCa cells and renders CXCR2+ NE cells resis- tant to hormonal therapy [androgen depri- vation therapy or enzalutamide treatment (ADT/Enza)]. Logistic regression analysis was performed using nonparametric Mann-Whitney U test; lines represent median and inter- quartile range. ns, nonsignificant; *P < 0.05.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Cell Culture, Colony Assay, Expressing, In Vivo, Binding Assay, ChIP-sequencing, Over Expression, CRISPR, Knock-Out, MANN-WHITNEY

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 4. CXCR2 expression in NE cells drives the secretion of proangiogenic factors and promotes the formation of premetastatic niche in the tumor environment. (A and B) Representa- tive images of immunofluorescence for CXCR2 (red) and vascular endothelial cell marker PECAM1 (green) in TMAs of human primary PCa are shown in (A). Nuclei (DAPI staining) are shown in blue. Spearman correlation between CXCR2 expression and blood vessel density in a panel of 77 PCa cases is shown in (B). (C and D) Representative IHC images (C) and quantification (D) of PECAM1 expression in LNCaP and LNCaP-CXCR2 xenograft tumors. (E to H) Representa- tive images (E) and quantification (F) of human cytokine antibody arrays of LNCaP or LNCaP-CXCR2. Representative images (G) and quantification (H) of human cyto- kine antibody arrays of C4-2B/MDVR- sgCtrl or C4-2B/MDVR-sgCXCR2. FBS, fetal bovine serum. (I to N) Representa- tive images (I) and quantification (J) of transwell Matrigel invasion assay of LNCaP and LNCaP-CXCR2 cells. The data were normalized to average in- vasion. Representative images (K) and quantification (L) of transwell Matrigel invasion assay of C4-2B/MDVR-sgCtrl and C4-2B/MDVR-sgCXCR2 cells. The data were normalized to average in- vasion. Representative images (M) and quantification (N) of spheroid invasion of C4-2B/MDVR-sgCtrl and C4-2B/ MDVR-sgCXCR2 cells at days 0 and 10, with leading edges indicated by the red arrows. Invasion distance normal- ized to day 0 distance for each sphere. (O) A model of CXCR2+ NE cells re- modeling the tumor microenviron- ment. MMP, matrix metalloproteinase. VEGF, vascular endothelial growth factor. Logistic regression analysis was performed using nonparametric Mann- Whitney U test; lines represent median and interquartile range. *P < 0.05.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Expressing, Immunofluorescence, Marker, Staining, Invasion Assay, MANN-WHITNEY

Journal: Science translational medicine

Article Title: Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

doi: 10.1126/scitranslmed.aax0428

Figure Lengend Snippet: Fig. 5. Advanced and therapy-resistant PCa is sensitive to CXCR2 inhibition. (A to C) Tumorigenesis of C4-2B/MDVR cells with/without CRISPR-Cas9 knockout of CXCR2. Representative images (A), quantification of colony-forming efficiency (B), and colony size (C) of C4-2B/MDVR cells with/without the CXCR2 gene deleted. Cells were cul- tured for 2 weeks. (D to F) Effect of na- varixin on enzalutamide-resistant PCa C4-2B/ MDVR cells’ growth in vivo. Images (D), weights (E), and volumes (F) of C4-2B/MDVR tumors in mice treated with navarixin (70 mg/kg) or vehicle control. (G) Representative images of IHC staining for AR and prostate-specific antigen in control and navarixin-treated C4-2B/MDVR xenografts. (H to J) Repre- sentative images (H) and quantification (I) of immunostaining for terminal deoxy- nucleotidyl transferase–mediated deoxy- uridine triphosphate nick end labeling (TUNEL) in tumors from mice treated with vehicle or CXCR2 inhibitor navarixin for 3 weeks. Representative images (J) of BCL2 associated X, apoptosis regulator (BAX) immunostaining of xenograft tumors in mice treated with/without navarixin for 3 weeks. (K and L) Representative images (K) and quantification (L) of blood vessel marker CD31 in mice treated with vehicle or navarixin (70 mg/kg) for 3 weeks. (M) Quantification of C4-2B tumor burden in mice treated with vehicle, enzalutamide, navarixin, or both enzalutamide and navarixin. (N and O) Quantification (N) and representative images (O) of immunostain- ing for TUNEL in mice treated with vehicle, enzalutamide, navarixin, or both enzalut- amide and navarixin. ANOVA, analysis of variance. Logistic regression analysis was performed using nonparametric Mann- Whitney U test; lines represent median and interquartile range.

Article Snippet: CXCR2 cDNA plasmid was synthesized by OriGene (SC321915).

Techniques: Inhibition, CRISPR, Knock-Out, In Vivo, Control, Immunohistochemistry, Immunostaining, End Labeling, TUNEL Assay, Marker, MANN-WHITNEY

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-CD182 conjugated to 147Sm (clone 5E8/CXCR2) , Fluidigm , Cat# 3147010B.

Techniques: Ubiquitin Proteomics, Recombinant, Multiplex Assay, Reverse Transcription, SYBR Green Assay, In Situ, shRNA, Software

Journal: Nature Cardiovascular Research

Article Title: Heart failure-specific cardiac fibroblasts contribute to cardiac dysfunction via the MYC–CXCL1–CXCR2 axis

doi: 10.1038/s44161-025-00698-y

Figure Lengend Snippet: a , Western blot analysis of CXCR2 expression in the negative control (NC), positive control (PC) and CM. Top: the NC is mouse embryonic fibroblasts, the PC is a human embryonic kidney cell line transfected with a Cxcr2 expression vector and the CM are cardiomyocytes of neonatal rats. Bottom: the NC is undifferentiated P19CL6 (mouse embryonic carcinoma) cells, the PC is a human embryonic kidney cell line transfected with a Cxcr2 expression vector and the CM are cardiomyocytes of mice. The experiment was repeated independently three times with similar results. b , Relative mRNA expression of HF markers in cardiomyocytes after addition of the medium conditioned by MYC overexpression in CFs, with and without a neutralizing antibody against CXCR2 ( n = 3 each). Data are shown as mean ± s.d. * P < 0.05; P < 0.001 (GFP ( n = 6) versus MYC ( n = 10), Nppa ), P = 0.0027 (MYC versus MYC plus anti-CXCR2 ( n = 5), Nppa ), P < 0.001 (GFP ( n = 7) versus MYC ( n = 10), Nppb ), P = 0.0027 (MYC versus MYC plus anti-CXCR2 ( n = 5), Nppb ), P < 0.001 (GFP ( n = 12) versus MYC ( n = 9), Myh7 ), P = 0.0040 (MYC versus MYC plus anti-CXCR2 ( n = 5), Myh7 ); significance was determined using the two-sided Mann–Whitney test for mean comparisons. c , Relative mRNA expression of HF markers measured by RT-qPCR in the hearts of control, TAC and TAC with neutralizing antibody against CXCR2. Data are shown as mean ± s.d. * P < 0.05; P < 0.001 (control ( n = 10) versus TAC ( n = 7), Nppa ), P = 0.017 (TAC versus TAC plus anti-CXCR2 ( n = 3), Nppa ), P = 0.0020 (control ( n = 10) versus TAC ( n = 4), Nppb ), P = 0.057 (TAC versus TAC plus anti-CXCR2 ( n = 3), Nppb ), P < 0.001 (control ( n = 10) versus TAC ( n = 6), Myh7 ), P = 0.038 (TAC versus TAC plus anti-CXCR2 ( n = 4), Myh7 ); significance was determined using the two-sided Mann–Whitney test for mean comparisons. d , Bar plots showing cardiac function evaluated by echocardiography in TAC mice with (anti-CXCR2) and without neutralizing antibody against CXCR2 (control). Data are shown as mean ± s.d. (TAC0w, n = 8 (control) and 7 (anti-CXCR2); TAC2w, 6 and 6; TAC4w, 6 and 6; TAC8w, 6 and 7; TAC12w, 8 and 7; TAC14w, 5 and 6; TAC16w, 7 and 6). * P < 0.05; P = 0.036 (TAC12w), P = 0.0037 (TAC14w), P = 0.015 (TAC16w); significance was determined using the two-sided Holm–Sidak test for mean comparisons. e , Comparison of cardiomyocyte size between control and TAC mice with and without neutralizing antibody against CXCR2 (anti-CXCR2). Cell size was evaluated using WGA staining. Quantitative analyses of the cell size in each mouse are shown ( n = 100 cells each). Box plots show the median (center line) and first and third quartiles (box edges), while the whiskers go from each quartile to the minimum or maximum. * P < 0.05; P < 0.001 (control versus TAC), P < 0.001 (TAC versus TAC + anti-CXCR2); significance was determined using the two-sided Mann–Whitney test for mean comparisons. f , Comparison of fibrotic areas between control and TAC mice with and without neutralizing antibody against CXCR2. Fibrosis was evaluated in the perivascular and interstitial regions by azan staining. Scale bars, 300 μm. Representative histological data and quantitative analyses of the fibrotic areas in each mouse are shown ( n = 7 each). Data are shown as mean ± s.d. * P < 0.05; P < 0.001 (control versus TAC, perivascular), P = 0.54 (TAC versus TAC plus anti-CXCR2, perivascular), P < 0.001 (control versus TAC, interstitial), P = 0.38 (TAC versus TAC plus anti-CXCR2, interstitial); significance was determined using the two-sided Mann–Whitney test for mean comparisons. g , Bar plots showing cardiac function evaluated by echocardiography in control, and shRNA of CXCR2-injected mice (shCXCR2) after TAC. Data are shown as mean ± s.d. ( n = 7 (control) and 5 (shCXCR2)). * P < 0.05; P = 0.025 (TAC8w); significance was determined using the two-sided Holm–Sidak test for mean comparisons. h , t -SNE plot of single-cell transcriptomes of human hearts (control: n = 1,673; HF: n = 29,496) (colored by cell clusters). Endo, endothelial cell; SM, smooth muscle cell; MC, myeloid cell; LC, lymphoid cell. i , t -SNE unsupervised clustering of fibroblasts in the human heart colored by cell clusters. j , Cell ratio of MYC -expressing CFs (control: n = 2; dilated cardiomyopathy (DCM): n = 4). Box plots show the median (center line) and first and third quartiles (box edges), while the whiskers go from each quartile to the minimum or maximum. k , Immunofluorescence for PDGFRα and smFISH for MYC and CXCL1 in human control hearts and DCM hearts. Scale bars, 20 μm. The experiment was repeated independently three times with similar results. The arrows indicate the colocalization of the MYC, CXCL1 and PDGFRα in the same cells. l , Bar plots showing the effect of CXCL1 on the contractile properties of iPSCM organoids. The plots show the rate of change of tissue contraction kinetics. Each parameter was normalized by the value of the control well ( n = 14 each). Data are shown as mean ± s.d. * P < 0.05; P = 0.047; significance was determined using the two-sided unpaired t -test for mean comparisons.

Article Snippet: Nitrocellulose membranes with proteins were washed with 0.05% Tween-20 in Tris-buffered saline (TBST), blocked (ImmunoBlock; CTKN001, KAC) for 30 min at room temperature (20–25 °C) and then incubated with primary antibody (goat polyclonal anti-GAPDH antibody (1:1,000; sc20357, Santa Cruz Biotechnology), rabbit monoclonal anti-MYC antibody (1:1,000; ab32072, Abcam), anti-CXCR2 antibody (1:1,000; PA1029; Boster Biological Technology), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (1:1,000; 9101S, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (137F5) antibody (1:1,000; 4695, Cell Signaling Technology) and CXCR2/CD182 Polyclonal Antibody (1:1000; Bioss)) at 4 °C overnight.

Techniques: Western Blot, Expressing, Negative Control, Positive Control, Transfection, Plasmid Preparation, Over Expression, MANN-WHITNEY, Quantitative RT-PCR, Control, Comparison, Staining, shRNA, Injection, Immunofluorescence

Journal: Nature Cardiovascular Research

Article Title: Heart failure-specific cardiac fibroblasts contribute to cardiac dysfunction via the MYC–CXCL1–CXCR2 axis

doi: 10.1038/s44161-025-00698-y

Figure Lengend Snippet: Related to Fig. . a : Experimental scheme of TAC mice injected with a neutralising antibody against CXCR2. b : Bar plots showing left ventricle chamber size and wall thickness measured by echocardiography in control and anti-CXCR2 mice after TAC. Mean and standard error are shown (TAC0w, n = 8 [control] and 7 [anti-CXCR2]; TAC2w, 6 and 6; TAC4w, 6 and 6; TAC8w, 6 and 7; TAC12w, 8 and 7; TAC14w, 5 and 6; TAC16w, 7 and 6). *, p < 0.05; p = 0.040 (LVDs, TAC16w); significance was determined using the two-sided Holm-Sidak test for mean comparisons. Source data are provided as a Source Data file. c : Bar plots showing left ventricle chamber size and wall thickness measured by echocardiography in control and shCXCR2 mice after TAC. Mean and standard error are shown (n = 6 [control] and 5 [shCXCR2] for TAC0w, TAC2w, TAC4w, TAC6w, TAC8w, and TAC10w). *, p < 0.05; p = 0.015 (LVDd, TAC10w), p = 0.0020 (LVDs, TAC10w); significance was determined using the two-sided Holm-Sidak test for mean comparisons. Source data are provided as a Source Data file. d : Bar plots showing cardiac function, left ventricle chamber size, and wall thickness measured by echocardiography in control and shRNA of Cxcx1 injected mice (shCXCL1) after TAC. Mean and standard error are shown (n = 7 [control] and 5 [shCXCL1] for TAC0w, TAC2w, TAC4w, TAC6w, TAC8w, and TAC10w). *, p < 0.05; p = 0.0064 (LVDd, TAC10w); significance was determined using the two-sided Holm-Sidak test for mean comparisons. Source data are provided as a Source Data file. e : Left panel, Western blot analysis of p-ERK, ERK, and GAPDH expression in cardiomyocytes from neonatal rats treated with CXCL1 alone or in combination with the anti-CXCR2 antibody. Representative data are shown. Right panel, quantification of Western blot optical densities, with p-ERK/ERK ratios displayed. *, p < 0.05; significance was determined using the two-sided Mann-Whitney test for mean comparisons. Source data are provided as a Source Data file.

Article Snippet: Nitrocellulose membranes with proteins were washed with 0.05% Tween-20 in Tris-buffered saline (TBST), blocked (ImmunoBlock; CTKN001, KAC) for 30 min at room temperature (20–25 °C) and then incubated with primary antibody (goat polyclonal anti-GAPDH antibody (1:1,000; sc20357, Santa Cruz Biotechnology), rabbit monoclonal anti-MYC antibody (1:1,000; ab32072, Abcam), anti-CXCR2 antibody (1:1,000; PA1029; Boster Biological Technology), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (1:1,000; 9101S, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (137F5) antibody (1:1,000; 4695, Cell Signaling Technology) and CXCR2/CD182 Polyclonal Antibody (1:1000; Bioss)) at 4 °C overnight.

Techniques: Injection, Control, shRNA, Western Blot, Expressing, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Heart failure-specific cardiac fibroblasts contribute to cardiac dysfunction via the MYC–CXCL1–CXCR2 axis

doi: 10.1038/s44161-025-00698-y

Figure Lengend Snippet: Related to Fig. . a : Immunofluorescence for PDGFRα and single-molecule fluorescence in situ hybridization for MYC and CXCL1 in human dilated phase HCM (dHCM) and sarcoidosis hearts. Scale bars, 20 μm. Arrows indicate the colocalization of the MYC, CXCL1, and PDGFRα in the same cells. b : CXCR2 expression data using single-cell RNA-seq from our previous report. Horizontal axis, TNNT2 expression; vertical axis, CXCR2 expression. Black squares, cardiomyocytes from human control hearts; blue triangles, cardiomyocytes from human hearts of dilated cardiomyopathy. c : Western blot analysis of CXCR2 and GAPDH expression in iPSCM. d : Relative mRNA expression of HF markers in iPSCM (n = 3–5 each). Data are shown as mean ± SD. *, p < 0.05; p = 0.029 (Nppa), p = 0.036 (Nppb); significance was determined using the two-sided Mann-Whitney test for mean comparisons. Source data are provided as a Source Data file.

Article Snippet: Nitrocellulose membranes with proteins were washed with 0.05% Tween-20 in Tris-buffered saline (TBST), blocked (ImmunoBlock; CTKN001, KAC) for 30 min at room temperature (20–25 °C) and then incubated with primary antibody (goat polyclonal anti-GAPDH antibody (1:1,000; sc20357, Santa Cruz Biotechnology), rabbit monoclonal anti-MYC antibody (1:1,000; ab32072, Abcam), anti-CXCR2 antibody (1:1,000; PA1029; Boster Biological Technology), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (1:1,000; 9101S, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (137F5) antibody (1:1,000; 4695, Cell Signaling Technology) and CXCR2/CD182 Polyclonal Antibody (1:1000; Bioss)) at 4 °C overnight.

Techniques: Immunofluorescence, Fluorescence, In Situ Hybridization, Expressing, RNA Sequencing, Control, Western Blot, MANN-WHITNEY

Journal: Nature Cardiovascular Research

Article Title: Heart failure-specific cardiac fibroblasts contribute to cardiac dysfunction via the MYC–CXCL1–CXCR2 axis

doi: 10.1038/s44161-025-00698-y

Figure Lengend Snippet: FACS analyses of CD3, CD11b, and Ly6G expression in mice hearts of sham (TAC0w), TAC4w, TAC8w, TAC12w, and TAC 12w with neutralising antibody against CXCR2.

Article Snippet: Nitrocellulose membranes with proteins were washed with 0.05% Tween-20 in Tris-buffered saline (TBST), blocked (ImmunoBlock; CTKN001, KAC) for 30 min at room temperature (20–25 °C) and then incubated with primary antibody (goat polyclonal anti-GAPDH antibody (1:1,000; sc20357, Santa Cruz Biotechnology), rabbit monoclonal anti-MYC antibody (1:1,000; ab32072, Abcam), anti-CXCR2 antibody (1:1,000; PA1029; Boster Biological Technology), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (1:1,000; 9101S, Cell Signaling Technology), p44/42 MAPK (Erk1/2) (137F5) antibody (1:1,000; 4695, Cell Signaling Technology) and CXCR2/CD182 Polyclonal Antibody (1:1000; Bioss)) at 4 °C overnight.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Pro-inflammatory role of neutrophils populations in trauma patients: monitoring neutrophil populations

doi: 10.3389/fimmu.2025.1565606

Figure Lengend Snippet: CD marker expression for individual neutrophil subpopulations. Cell surface expression of neutrophil markers on different neutrophil subpopulations at TP1, including (A) CD16, (B) CD62L, (C) CD66b, (D) CD11b, (E) CD10, (F) CD181, and (G) CD182. CD16 low CD62L low neutrophils exhibit low expression of all analyzed markers. Decreased expression of CD10 and CD11b on banded neutrophils (BN). Data were non-normally distributed (Shapiro-Wilk test), and statistical analysis was conducted using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (all vs. all); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample size CD16, CD62L CD66b and CD11b: TP1: N = 33 Non-SIRS patients, 16 SIRS patients; CD10: TP1: N = 32 Non-SIRS patients, 9 SIRS patients; CD181: TP1: N = 39 Non-SIRS patients, 11 SIRS patients; CD182: TP1: N = 31 Non-SIRS patients, 12 SIRS patients.

Article Snippet: Additional antibodies included CD181 APC (clone 8F1/CXCR1), CD182 APC (clone 5E8/CXCR2), and CD66b Alexa Fluor 700 (clone 10F5; Sony Biotechnology, San Jose, CA, USA).

Techniques: Marker, Expressing