Journal: Journal of Neuroinflammation

Article Title: Differential neuro-immune patterns in two clinically relevant murine models of multiple sclerosis

doi: 10.1186/s12974-019-1501-9

Figure Lengend Snippet: Quantitative analysis of CXCL13, Chi3l4, and IgG1 expression in spinal cord samples from TMEV-IDD and R-EAE mice. Both RT-qPCR for mRNA and ELISA for protein of CXCL13 ( a , d ) and IgG1 ( c , f ) showed significant increases in expression in TMEV-IDD mice vs . both R-EAE and control mice. Conversely, both RT-qPCR for mRNA ( b ) and ELISA for protein ( e ) of Chi3l4 showed increased expression in R-EAE mice vs . both TMEV-IDD and control mice. RT-qPCR results are representative of 3 independent experiments ( n = 31) for TMEV-IDD and 3 independent experiments ( n = 16) for R-EAE. Likewise, ELISA results are representative of 3 independent experiments ( n = 15) for TMEV-IDD and 2 independent experiments ( n = 10) for R-EAE. Since the effect for age was not significant, sham mice were analyzed as all from one population ( n = 14 for mRNA and n = 8 for proteins). Data are shown as mean ± SEM. **** p < 0.0001; * p < 0.05

Article Snippet: Specific proteins levels were then measured by using commercially available ELISAs for the quantification of Chi3l4 (LSBio, Seattle, WA) and CXCL13 (BioLegend, San Diego, CA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Differential neuro-immune patterns in two clinically relevant murine models of multiple sclerosis

doi: 10.1186/s12974-019-1501-9

Figure Lengend Snippet: Chemokine levels (pg/mL) in CSF and serum of TMEV-IDD mice, R-EAE mice, and sham controls. The levels of CXCL13, CXCL12, and CCL19 were measured in individual CSF ( a , c , e ) and serum ( b , d , f ) specimens by Luminex bead immunoassays. CSF chemokine levels were higher in TMEV-IDD mice as compared to both R-EAE mice and sham controls. Conversely, serum chemokine levels were higher in R-EAE as compared to both TMEV-IDD mice and sham controls. Data are representative of 3 independent experiments ( n = 11) for TMEV-IDD and 1 experiment ( n = 6) for R-EAE. Since the effect for age was not significant, sham mice were analyzed as all from one population ( n = 5). Data are shown as mean ± SEM. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: Specific proteins levels were then measured by using commercially available ELISAs for the quantification of Chi3l4 (LSBio, Seattle, WA) and CXCL13 (BioLegend, San Diego, CA).

Techniques: Luminex

Journal: The Journal of Experimental Medicine

Article Title: Follicular dendritic cells help establish follicle identity and promote B cell retention in germinal centers

doi: 10.1084/jem.20111449

Figure Lengend Snippet: DTx-mediated selective FDC ablation disrupts B cell follicle architecture despite retention of CXCL13. Unimmunized CD21-DTR chimeras were control (Ctrl) or DTx treated 2 d before analysis of spleen (spl) and pLN sections. (A) Immunofluorescence analysis for BP3 + stromal cells, CD35 + FDCs, and IgD + follicular B cells. (B) In situ hybridization for Cxcl13 mRNA with serial sections stained for B220 and CD35 or for Cxcl13 and CD35. (C) Quantitative RT-PCR analysis for Cxcl13 , Cr1 (CD35), Mfge8 , and Tnsf13B (BAFF) mRNA in pLNs, spleen, and mLNs, normalized to Hprt mRNA. Data are representative of four experiments (mean ± SEM). Statistical analysis was performed with the two-tailed unpaired Student’s t test. *, P < 0.05; n.s., not significant. (D) Immunohistochemical analysis for CXCL13 and CD3 or B220 in serial sections of spleen and pLNs. Data in A, B, and D are representative of at least three experiments (at least two mice of each type per experiment). Bars: (A [left] and B [left and middle]) 200 µm; (A and D, right) 100 µm; (B [right] and D [left]) 50 µm.

Article Snippet: Cryosections of 7 µm were fixed and stained immunohistochemically as previously described ( ) with the following first antibodies: PE-conjugated anti-IgD (11-26c.2a; BD), biotin-conjugated anti-CD35 (8C12; BD), rat anti–mouse fibroblast (ER-TR7; Novus Biologicals), rabbit anti–mouse Collagen IV (Abcam), rat anti–mouse B220 (RA-6B2; BD), biotin-conjugated anti–T/B cell activation antigen or Ly77 (GL7; BD), biotin conjugated anti–mouse CD3e (145-2C11; BD), goat anti–mouse CXCL13 (R&D Systems), goat anti–mouse CCL21 (R&D Systems), rat anti-CD169 (Ser4; P. Crocker, University of Glasgow, Glasgow, Scotland, UK), rat anti–mouse madcam-1 (MECA-367; BD), rat anti–mouse FDC (FDC-M1; BD), and FITC-conjugated rat anti–mouse CD16/32 (FcγRII/III; UCSF Hybridoma Core).

Techniques: Immunofluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR, Two Tailed Test, Immunohistochemical staining

Journal: eBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: The depletion of hMENA 11a down-regulates LTβR and up-regulates FN1. In node-negative patients with NSCLC, hMENA 11a expression associates with intratumoral TLS . a. Volcano plots showing differentially expressed genes (q-value <0.05, n = 1096, GSE217451) in Si-hMENA 11a versus control H1650 NSCLC cells (n = 3). Reported are the negative log10-transformed adjusted P values plotted against the log2 fold changes. Dots represent individual genes. b . qRT-PCR analysis of LTβR mRNA expression in the indicated cell lines transfected with control (Si-CNTR), and hMENA 11a pool SiRNAs (Si-hMENA 11a ) or with plasmid control (CNTR) and hMENA 11a expressing vector (hMENA 11a ). The control of hMENA 11a expression in the transfected cells by WB is reported in Supplementary Figure S3a . Data are reported as the mean ± SD of technical triplicates which are representative of two independent experiments. P value was calculated by 2-tailed Student's t test. c. qRT-PCR analysis showed that LTβR and hMENA 11a mRNA expression level correlate in NSCLC cell lines. The value of Spearman correlation is reported. d. Consecutive sections of a representative NSCLC primary tumor hMENA 11a positive, showing low stromal FN1, TLS IT localization and high CXCL13. Magnification 8×. Scale bar 300 μm. Right, histograms relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA 11a positive cases more frequently show TLS within the tumor area (TLS IT). e. Consecutive sections of a representative case of lung adenocarcinoma hMENA 11a negative, showed high stromal FN1, TLS PT and low CXCL13. Right, histograms are relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA 11a negative (hMENA 11a low /hMENA (t) high ) cases more frequently show peritumoral TLS (TLS PT). P value was estimated with Fisher Exact test.

Article Snippet: CXCL13 detection was obtained by the use of human CXCL13 Antibody (AF801 R&D Systems).

Techniques: Expressing, Control, Transformation Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: eBioMedicine

Article Title: Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

doi: 10.1016/j.ebiom.2024.105003

Figure Lengend Snippet: hMENA/hMENAΔv6 influences the expression and signaling of LTβR and the secretion of CXCL13 in CAFs. The ‘epithelial’ hMENA 11a isoform in tumor cells affects CXCL13 production by T RM cells . a. qRT-PCR analysis of LTβR mRNA expression in the CAFs obtained from four different patients with NSCLC (#359, #358, #391, #405), transfected with control (Si-CNTR), and hMENA(t) pool siRNAs (Si-hMENA(t)). Data reported are the mean of technical triplicates. P value of paired 2-tailed Student's t test is reported. b. Representative WB analysis with the indicated Abs of protein extracts from CAF #302 transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA, untreated or treated with 50 ng/mL of LIGHT for 24 h. Fold change of P-p65/p65 and of p52/p100 staining intensity (right). Data reported are the mean of 4 different experiments ± SD. Adjusted P values of One-way ANOVA followed by Tukey's multiple comparisons procedures are reported when significant. c. CXCL13 production evaluated by ELISA assay of CAFs derived from three different patients (#302; #358; #571) transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA. Data reported are the mean of technical triplicates of pg/mL normalized for total protein content in three biological replicates. P value of paired two tailed t test, is reported d. Percentage of CXCL13 chemokine, evaluated by multiparametric flow cytometry, in ex-vivo TILs isolated from eight patients with NSCLC, after culture (24 h) with CM from H1650 tumor cells silenced for hMENA 11a (Si-hMENA 11a ), or control (Si-CNTR). Results within CD8 + T RM (CD103 + CD69 + , left panel) or CD4 + T RM (CD103 + CD69 + , right panel) are shown. Statistical significance was determined using non-parametric Wilcoxon rank test.

Article Snippet: CXCL13 detection was obtained by the use of human CXCL13 Antibody (AF801 R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Staining, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test, Flow Cytometry, Ex Vivo, Isolation

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: Cxcl13 - and Calca -mRNAs are present in the murine tracheal epithelium. RT-PCR experiments with cDNA obtained from tracheal epithelium of two C57BL/6RJ animals using primers for Cxcl13 (250 bp), Calca (158 bp), and β-actin (300 bp). Amplicons of Calca , Cxcl13 , and β-actin are detected in both tracheal epithelium samples (TE). Controls = RNA samples processed without reverse transcriptase (-RT) and water (H 2 O) without adding cDNA

Article Snippet: Floating sections were rinsed in PBS, and unspecific protein binding sites were saturated with 10% normal porcine serum in 0.005 M PBS for 1 h. Sections were incubated overnight with goat polyclonal antibody against CXCL13 (1:400 AF470, R&D Systems) or rabbit polyclonal antibody against αCGRP (1:20,000; T-4032, Peninsula Laboratories) followed by incubation for 1 h with peroxidase-conjugated pig anti-rabbit Ig (1:100; P0217, Dako, Santa Clara, USA) to detect CGRP or with biotinylated secondary donkey anti-goat IgG (1:400; 705–065-147, Dianova) to detect CXCL13.

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13-positive and neuroendocrine cells are unevenly distributed in the trachea. a – d Tracheal whole mount immunohistochemistry, CLSM, with antibodies against CXCL13 (green) and CGRP (red), labeling single neuroendocrine cells and nerve fibers. The density of neuroendocrine (CGRP-positive) cells, CXCL13-immunoreactive cells, and nerve fibers is higher between the cartilage rings (Ca). The density of neuroendocrine cells and CXCL13-immunoreactive cells decreases from cranial (larynx) ( b ) to caudal (bifurcation) ( d ); each channel is individually shown in Supplementary Fig. 1 . Numerous CGRP-positive nerve fibers ( <) are visible throughout the whole trachea. Maximum intensity projection of z-stack of confocal optical sections. e, f Cell densities (mean ± SEM) quantified on tracheal whole mounts double-labelled for CXCL13 and PGP9.5 (neuroendocrine cell marker). Immunoreactive cells dominate in intercartilage regions ( e ), and their density continuously declines along the cranio-caudal axis ( f ). In f , counts include both the area overlying a cartilage and the next intercartilage region; colour coding along the cranio-caudal axis identifies data from the same trachea. g RT-qPCR. Cxcl13 expression is about 3 times higher in tracheal rings 1–3 compared to rings 8–10

Article Snippet: Floating sections were rinsed in PBS, and unspecific protein binding sites were saturated with 10% normal porcine serum in 0.005 M PBS for 1 h. Sections were incubated overnight with goat polyclonal antibody against CXCL13 (1:400 AF470, R&D Systems) or rabbit polyclonal antibody against αCGRP (1:20,000; T-4032, Peninsula Laboratories) followed by incubation for 1 h with peroxidase-conjugated pig anti-rabbit Ig (1:100; P0217, Dako, Santa Clara, USA) to detect CGRP or with biotinylated secondary donkey anti-goat IgG (1:400; 705–065-147, Dianova) to detect CXCL13.

Techniques: Immunohistochemistry, Labeling, Marker, Quantitative RT-PCR, Expressing

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13 is restricted to neuroendocrine cells in the tracheal epithelium. a , b High-resolution of tracheal whole mount immunohistochemistry, labeling CXCL13 + cells in green and CGRP + cells and nerve fibers in red, revealed CXCL13 and CGRP colocalization within the same cell. Inset in a shows the magnified region of the labeled cell with only limited overlap of immunoreactivites (scale bar 1 µm). Images were acquired using a confocal laser scanning microscope (FLUOVIEW FV3000; Olympus), single confocal optical section. b CXCL13 + cell in contact to a CGRP + nerve fiber ( <). Maximum intensity projection of z-stack of confocal optical sections. c Immunohistochemistry of a tracheal cryosection from a ChAT-eGFP animal. In the tracheal epithelium, single cells are double-stained with antibodies against PGP9.5 and CXCL13 (arrowhead) or PGP9.5 only (arrow). An eGFP-positive cell (asterisk) is not labeled with antibodies against CXCL13 or PGP9.5. PGP9.5 + nerve fibers are in contact to PGP9.5 + epithelial cells. d Triple-immunofluorescence of tracheal cryosection shows co-labeling of single epithelial cells for CXCL13 (green), CGRP (yellow), and PGP9.5 (red) (arrowheads). A single PGP9.5-labeled cell with neither CXCL13 nor CGRP immunolabeling is also present (asterisk). e–f Transmission electron microscopy. e Ultrastructure of a tracheal neuroendocrine cell (NEC) with a pyramidal or flask-like shape and a small apical part reaching the lumen with microvilli. e′ Higher magnification of the basal part, showing the presence of numerous dense core vesicles (DCV). f Ultrastructural immunohistochemistry with antibodies against CXCL13 shows an immunoreactive cell with the diffuse DAB reaction product. f′ Higher magnification of the basal part, showing the presence of numerous DCV

Article Snippet: Floating sections were rinsed in PBS, and unspecific protein binding sites were saturated with 10% normal porcine serum in 0.005 M PBS for 1 h. Sections were incubated overnight with goat polyclonal antibody against CXCL13 (1:400 AF470, R&D Systems) or rabbit polyclonal antibody against αCGRP (1:20,000; T-4032, Peninsula Laboratories) followed by incubation for 1 h with peroxidase-conjugated pig anti-rabbit Ig (1:100; P0217, Dako, Santa Clara, USA) to detect CGRP or with biotinylated secondary donkey anti-goat IgG (1:400; 705–065-147, Dianova) to detect CXCL13.

Techniques: Immunohistochemistry, Labeling, Laser-Scanning Microscopy, Staining, Immunofluorescence, Immunolabeling, Transmission Assay, Electron Microscopy

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13 is expressed in two-thirds of the murine tracheal neuroendocrine cells. a , b Immunohistochemistry of tracheal whole mounts and the corresponding quantification of their immunoreactive cells; maximum intensity projections of z-stacks of confocal optical sections. a Immunohistochemistry with antibodies against CXCL13 ( a ) (green) and PGP9.5 ( a′ ) (red), labeling single neuroendocrine cells and nerve fibers. CXCL13 + /PGP9.5 + cells are indicated by arrowheads; CXCL13 − /PGP9.5 + cells are indicated by ( <). Data points in the scatter plot ( a‴ ) represent mean values of counts in one trachea ( n = 5 tracheas); mean and SEM are indicated. The pie chart shows the percentages of CXCL13 + /PGP9.5 + and CXCL13 − /PGP9.5 + cells ( n = 2254 cells pooled from 5 tracheas). b Immunohistochemistry with antibodies against CXCL13 ( b ) (green) and CGRP ( b′ ) (red), labeling single neuroendocrine cells and nerve fibers. CXCL13 + /CGRP + cells are indicated by arrowheads; CXCL13 − /CGRP + cells are indicated by ( <); CXCL13 + /CGRP − cells are indicated by (*). Data points in the scatter plot ( b‴ ) represent mean values of counts in one trachea ( n = 5 tracheas); mean and SEM are indicated. The pie chart shows the percentages of phenotypes ( n = 2650 cells pooled from 5 tracheas)

Article Snippet: Floating sections were rinsed in PBS, and unspecific protein binding sites were saturated with 10% normal porcine serum in 0.005 M PBS for 1 h. Sections were incubated overnight with goat polyclonal antibody against CXCL13 (1:400 AF470, R&D Systems) or rabbit polyclonal antibody against αCGRP (1:20,000; T-4032, Peninsula Laboratories) followed by incubation for 1 h with peroxidase-conjugated pig anti-rabbit Ig (1:100; P0217, Dako, Santa Clara, USA) to detect CGRP or with biotinylated secondary donkey anti-goat IgG (1:400; 705–065-147, Dianova) to detect CXCL13.

Techniques: Immunohistochemistry, Labeling

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: In silico analysis of single-cell mRNA sequencing data revealed CXCL13 expression predominantly in neuroendocrine cells of the tracheal epithelium. a – c In silico analysis of published sequencing data (GSE102580) of murine tracheal epithelial cells (Plasschaert et al. ). a SPRING plot (Uniform Manifold Approximation and Projection, UMAP) shows eight distinct cell clusters, namely basal, secretory, Krt4/13 + , ciliated, solitary cholinergic chemosensory (brush/tuft), cycling basal and solitary neuroendocrine cells, and ionocytes. b SPRING and violin plots showing that Cxcl13 -mRNA is predominantly expressed within the neuroendocrine cell cluster. c Heat map shows the most differentially expressed genes (fold change > 1) between CXCL13 + and CXCL13 − neuroendocrine cells among the 200 highest expressed genes within the neuroendocrine cell cluster and typical neuroendocrine cell marker genes (e.g., Ascl1 , Uchl1 , Calca , Chga , Chgb )

Article Snippet: Floating sections were rinsed in PBS, and unspecific protein binding sites were saturated with 10% normal porcine serum in 0.005 M PBS for 1 h. Sections were incubated overnight with goat polyclonal antibody against CXCL13 (1:400 AF470, R&D Systems) or rabbit polyclonal antibody against αCGRP (1:20,000; T-4032, Peninsula Laboratories) followed by incubation for 1 h with peroxidase-conjugated pig anti-rabbit Ig (1:100; P0217, Dako, Santa Clara, USA) to detect CGRP or with biotinylated secondary donkey anti-goat IgG (1:400; 705–065-147, Dianova) to detect CXCL13.

Techniques: In Silico, Sequencing, Expressing, Marker

Journal: Cell and Tissue Research

Article Title: CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

doi: 10.1007/s00441-021-03552-2

Figure Lengend Snippet: CXCL13 is less expressed in murine broncho-pulmonary solitary and clustered neuroendocrine cells. Immunohistochemistry of lung cryosections with antibodies against CXCL13 (orange) and CGRP (green) and the relative frequencies of immunoreactive phenotypes. a Solitary neuroendocrine cell co-labeled with antibodies against CXCL13 and CGRP. b Solitary neuroendocrine cell only labeled with antibodies against CGRP. c A cluster of neuroendocrine cells (neuroepithelial body) consisting of more than 7 cells, 2 of them are co-labeled with antibodies against CXCL13 and CGRP. d Pie chart shows percentages of CXCL13 + /CGRP + and CXCL13 − /CGRP + -immunolabeled cells in solitary neuroendocrine cells ( n = 73 cells pooled from 5 animals). e Pie chart shows the percentage of CXCL13 + /CGRP + and CXCL13 − /CGRP + -immunolabeled cells in neuroepithelial bodies ( n = 1475 cells pooled from 5 animals)

Article Snippet: Floating sections were rinsed in PBS, and unspecific protein binding sites were saturated with 10% normal porcine serum in 0.005 M PBS for 1 h. Sections were incubated overnight with goat polyclonal antibody against CXCL13 (1:400 AF470, R&D Systems) or rabbit polyclonal antibody against αCGRP (1:20,000; T-4032, Peninsula Laboratories) followed by incubation for 1 h with peroxidase-conjugated pig anti-rabbit Ig (1:100; P0217, Dako, Santa Clara, USA) to detect CGRP or with biotinylated secondary donkey anti-goat IgG (1:400; 705–065-147, Dianova) to detect CXCL13.

Techniques: Immunohistochemistry, Labeling, Immunolabeling

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Novel biomarker candidates for OSCC identified using microarray analysis.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Biomarker Discovery, Microarray, Binding Assay

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Expression of CXCL13 mRNA in primary OSCC tissues by RT‐qPCR. (A) CXCL13 expression was higher in tumor tissues compared to adjacent normal tissues in all cases. (B) Significant upregulation of CXCL13 mRNA was observed in tumor tissues. ** p < 0.01 compared to adjacent normal tissues.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum levels of CXCL13 protein in patients with OSCC. (A) The average serum CXCL13 protein in 125 patients with OSCC was 72.8 ρg/mL. (B) The average serum CXCL13 protein in healthy individuals was 33.5 ρg/mL. (C) The reference value determined by the ROC curve for maximizing the TPF/FPF ratio was 53.0 ρg/mL. FPF, false‐positive fraction; TPF, true‐positive fraction.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 levels in patients with OSCC.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Diagnostic sensitivity of serum CXCL13 and SCC in patients with OSCC.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Diagnostic Assay

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 and clinicopathological factors in patients with OSCC. (A) Serum CXCL13 protein levels in T classification. The serum CXCL13 proteins increased in accordance with primary tumor size. The serum CXCL13 levels in T1 were significantly higher than in T3 and T4. (B) The serum CXCL13 proteins in recurrence. The serum levels of CXCL13 proteins in patients with recurrence were significantly elevated. (C) Patients with recurrence were classified into primary recurrence, neck LNM, and distant metastasis (including multiple responses). The serum levels of CXCL13 proteins in neck LNM were significantly elevated. * p < 0.05, ** p < 0.01.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 and prognosis in patients with OSCC. A comparison of overall survival (OS) and disease‐free survival (DFS) in patients with OSCC was made among the two groups, which were classified based on the median serum CXCL13 level using the Kaplan–Meier method with a log‐rank test. High expression of serum CXCL13 indicated poor prognosis in both OS (A, p = 0.044) and DFS (B, p = 0.018) across all stages. In Stage I/II, the groups had no significant difference in OS (C, p = 0.696) and DFS (D, p = 0.435). In Stage III/IV, both OS (E, p = 0.020) and DFS (F, p = 0.009) were significantly poorer in patients with high CXCL13 levels.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Comparison, Expressing

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Visium spatial transcriptome analysis using two cases of primary OSCC tissues. (A) Case 1: Primary tongue SCC tissues were classified into eight clusters based on the gene expression profile. Among these, the tumor area comprised four clusters (Cluster 1, 3, 5, and 6). CXCL13 expression was upregulated in three of four clusters within the tumor area. CD8 expression was detected in all tumor clusters. (B) Case 2: In Case 2, primary tongue SCC tissues were classified into seven clusters. The tumor area consisted of four clusters (Clusters 1, 2, 3, and 6). The expression of CXCL13 and CD8 was upregulated in three of four clusters within the tumor area. CD4 expression was detected only in Cluster 2 in the tumor area but not in Case 1.

Article Snippet: TaqMan probes and primers for CXCL13 (Hs00757930_m1) and HMBS (Hs00609297_m1) were obtained from Thermo Fisher Scientific.

Techniques: Gene Expression, Expressing

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) A PCR array was used to detect the expression of 84 cytokines/chemokines in eight highly polluted region (HPR) lung cancers. ( B ) The ratios of CXCL13 in tumor samples to their counterpart normal lung tissues from both the HPR and control region (CR) non-small cell lung cancers (NSCLCs). ( C ) Comparison of the CXCL13 tumor /CXCL13 normal values of the HPR patients with the CR cases. ( D, E ) CXCL13 expression was detected by immunohistochemistry (IHC) in HPR and CR patients ( D ), and the immunoreactivity score was calculated ( E ). ( F ) Western blot analyses of lysates from the tumors and adjacent normal lung tissues harvested from CR NSCLCs. ( G ) The concentrations of CXCL13 in the blood samples from healthy donors (HDs) and HPR and CR patients were detected by ELISA. ( H, I ) CXCL13 expression in Oncomine reports. ( H ) CXCL13 expression was detected by microarrays in tumor samples and normal lung tissues. AD, adenocarcinoma; A+S, adenocarcinoma and squamous cell carcinoma; Ca, Canada; It, Italy; Jp, Japan; SC, squamous cell carcinoma; Tw, Taiwan, China. ( I ) The expression of CXCL13 was detected in tumor tissues of smokers and non-smokers. ( J ) In mouse Gene Expression Omnibus (GEO) data sets, the expression of cxcl13 in indicated mice was detected by microarray. ( K ) The relationship between the CXCL13 expression and the tumor stages of lung cancer patients. ( L ) Overall survival of 54 CR patients (see for their baseline demographic characteristics). The median follow-up was 1087 days (range, 187–1845 days). DOI: http://dx.doi.org/10.7554/eLife.09419.003 10.7554/eLife.09419.004 Figure 1—source data 1. Sequences of primers for real-time PCR and ChIP, and siRNA. DOI: http://dx.doi.org/10.7554/eLife.09419.004

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay, Microarray, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: Baseline demographic characteristics of 54 control region (CR) lung cancer patients whose survival information was available. DOI: http://dx.doi.org/10.7554/eLife.09419.008

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques:

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: Baseline demographic characteristics of the 201 patients who underwent CXCL13 analyses. DOI: http://dx.doi.org/10.7554/eLife.09419.006

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques:

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: Multivariate logistic analyses of the association between CXCL13 high expression and clinical characteristics. DOI: http://dx.doi.org/10.7554/eLife.09419.007

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Expressing

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) A PCR array analysis of the expression of 84 cytokines/chemokines in 16HBE normal lung epithelial cells treated with 1 μM BaP for 30 days. ( B ) The cells were treated with BaP at 10 μM for indicated time points or with the indicated concentrations for 72 hr, and CXCL13 expression was assessed by real-time RT-PCR. The experiments were conducted in triplicate and repeated three times. The error bars represent the SD. ( C ) The cells were treated with BaP as described in ( B ), and the concentration of CXCL13 in the supernatants was evaluated by ELISA. ( D ) The A/J mice were treated with BaP and/or dexamethasone (DEX) for 5 weeks (see also ) and sacrificed 6 months later. The lung tissues were isolated and analyzed by Hematoxylin and eosin (HE) staining or immunohistochemistry (IHC) using an anti-Cxcl13 antibody (left panel). The immunoreactivity score was calculated (right panel). ( E ) Cxcl13 expression was detected in the lung tissues by real-time PCR. ( F ) The concentration of Cxcl13 in mouse serum was assayed by ELISA. ( G ) IHC assays of mice’ lung tumor tissues using anti-Cd68, anti-Ttf1, and anti-Cxcl13 antibodies. ( H ) Immunofluorescence assay of mice’ lung tumor tissues using antibodies against Cxcl13 (green), Cd68 (red), and Ttf1 (white). 4',6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus (blue). ( I ) The survival curves of the mice treated with BaP and/or DEX (n=8 for each group). DOI: http://dx.doi.org/10.7554/eLife.09419.009

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) The AhR binding site is located at 1.7 kb downstream of the CXCL13 transcription start site (TSS). ( B ) A chromatin immunoprecipitation (ChIP) assay was performed in BaP-treated or untreated 16HBE cells. The enriched CXCL13 was detected by qPCR. ( C ) The A549 cells were transfected with the wild-type (WT) or mutant (deletion mutation (mut) in the XRE-like sequence) CXCL13 promoter-luciferase reporter construct, treated with BaP and/or α-NF for 48 hr, and assessed by the luciferase assays. ( D, E ) A549 cells were transfected with AhR-specific siRNAs, and western blot was performed to detect the expression of AhR. Three siRNAs were used, and the result of one was shown ( D ). Luciferase assays were performed in A549 cells transfected with the WT CXCL13 promoter-luciferase reporter construct and siRNAs in the absence or presence of BaP ( E ). ( F, G ) Western blot analyses of AhR in the cytoplasm and nucleus of 16HBE cells co-incubated with BaP, with or without α-NF treatment ( F ) or siRNA transfections ( G ). ( H, I ) Immunofluorescence assays of AhR expression in 16HBE cells co-incubated with BaP, with or without α-NF treatment ( H ) or siRNA transfections ( I ). ( J, K ) CXCL13 mRNA (detected by qPCR; J ) and protein (in supernatants of the cells detected by ELISA; K ) levels in the AhR-silenced 16HBE cells treated with BaP and/or α-NF. DOI: http://dx.doi.org/10.7554/eLife.09419.011

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Transfection, Mutagenesis, Sequencing, Luciferase, Construct, Western Blot, Expressing, Incubation, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) The expression of Cxcl13 in the mice. (B) Schematic represents the protocols for administration of BaP in the mice. ( C ) The expression of Cxcr5 in the mice. DOI: http://dx.doi.org/10.7554/eLife.09419.013

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Expressing

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) Cxcl13 deficiency mice were treated with BaP, sacrificed 120 days, 180 days or 240 days later, and the tumor nodules in histologic sections were analyzed. See also . ( B ) MicroCT scanning images and HE staining of lung sections from the BaP-treated Cxcl13 wild-type (WT) or knockout mice. ( C ) Tumor volume of the microCT scanning of the mice. ( D ) Serum concentrations of Cxcl13 in the BaP-treated Cxcl13 WT or knockout mice. ( E ) Life span of the BaP-treated Cxcl13 +/+ , Cxcl13 +/- , and Cxcl13 -/- mice. ( F, G ) Cxcr5 expression in non-small cell lung cancers (NSCLCs, n=24; F) and in A/J mice treated with BaP (n=6 for each group; G ). ( H ) Cxcr5 deficiency mice were treated with BaP, sacrificed 120 days, 180 days or 240 days later, and the tumor nodules in histologic sections were analyzed. See also . ( I ) MicroCT scanning images and HE staining of lung sections from BaP-treated Cxcr5 WT or knockout mice. ( J ) Tumor volume of the microCT scanning of the mice. ( K ) Life span of the BaP-treated Cxcr5 WT or knockout mice. *p<0.05; **p<0.01. DOI: http://dx.doi.org/10.7554/eLife.09419.012

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Staining, Knock-Out, Expressing

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A, B ) Flow cytometry analysis of Cd68 + macrophages in BaP-induced tumors. A representative gating is shown. The numbers indicate the Cd68 + cells in the quadrant expressed as the percentage of the total Cd45+ leukocytes from the same tumor ( A ). The means+SD of the Cd68 + cells from the mice (n=10 for each group) are shown ( B ). See also . ( C ) IHC analysis of CD68 + macrophages in tumor samples from BaP-treated mice and highly polluted region (HPR) patients. ( D ) Flow cytometry analysis of Cd68 + macrophages isolated from tumor samples of mice treated with 50 mg/kg BaP using an anti-Cxcr5 antibody. ( E ) Immunofluorescence analysis of tumor-associated macrophages in tumor samples from HPR patients and THP-1 cells using anti-CD68 and anti-CXCR5 antibodies; DAPI was used to counterstain the nucleus. ( F ) A trans-well migration assay was performed by plating THP-1 cells in the lower chambers, and the indicated cells in the upper chambers, with or without anti-CXCR5 antibody. ( G, H ) Bioluminescent assays of mice that were inoculated with A549- Luciferase (Luc) or A549-Luc-CXCL13 cells (8×10 5 ) in the right lung. THP-1 cells (8×10 5 ) were injected via the tail vein. Representative images ( G ) and total luminous flux ( H ) were shown. ( I ) Lung sections of the mice were stained with HE. DOI: http://dx.doi.org/10.7554/eLife.09419.014

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Flow Cytometry, Isolation, Immunofluorescence, Migration, Luciferase, Injection, Staining

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) The pathway analysis of CXCL13 associated genes. The data from the microarray data sets of the eight highly polluted region (HPR) lung cancers are shown. See also . ( B ) Gene ranking according to the correlation with CXCL13 expression. The genes were filtered based on extracellular localization to identify paracrine mediators. The list on the right shows the genes that correlate most significantly with CXCL13 . ( C ) The mRNA expression of the candidate gene was detected in THP-1 cells by qPCR. ( D ) The concentration of SPP1 in supernatants of THP-1 cells in the absence or presence of CXCL13. ( E ) Trans-well migration assays were performed by plating THP-1 cells (transfected with siSPP1 or siNC) in the lower chambers, and the indicated cells (CXCL13-treated or untreated) in the upper chambers. ( F ) Spp1 serum concentrations of mice treated with benzo(a)pyrene (BaP) and/or dexamethasone (DEX) were detected by ELISA. ( G ) Spp1 expression in lung section of mice treated with BaP and/or DEX was determined by IHC. ( H ) IHC assays using antibodies against Cd68, Ttf1, and Spp1. ( I ) Immunofluorescence assay using antibodies against Spp1 (green), Cd68 (red), and Ttf1 (white). DAPI was used to stain the nucleus (blue). DOI: http://dx.doi.org/10.7554/eLife.09419.016 10.7554/eLife.09419.017 Figure 6—source data 1. CXCL13 -associated genes in lung cancer. DOI: http://dx.doi.org/10.7554/eLife.09419.017

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Microarray, Expressing, Concentration Assay, Migration, Transfection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: eLife

Article Title: The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

doi: 10.7554/eLife.09419

Figure Lengend Snippet: ( A ) The expression of indicated genes in lung tissues from A/J mice treated with BaP and/or dexamethasone (DEX) was detected by real-time PCR. ( B ) Western blot analyses of lysates of lung tissues from A/J mice treated with BaP and/or DEX. ( C ) Western blot analyses of cytoplasmic and nucleic protein fractions of lung tissues from A/J mice treated with BaP and/or DEX, using indicated antibodies. ( D ) Western blot analysis of lysates of lung tissues from NOD/SCID mice injected with indicated cells. ( E ) Western blot analyses of cytoplasmic and nucleic protein fractions in lung tissues from NOD/SCID mice injected with indicated cells. ( F ) Western blot analyses of lysates of lung tissues from Cxcr5 mutant mice treated with BaP. ( G ) IHC assays for the expression of β–catenin in Cxcr5 mutant mice upon BaP. ( H ) Immunofluorescence analyses of β-catenin in A549 cells transfected with control vector (V) or SPP1, or treated with THP-1 supernatant (Mϕs.) or supernatant of THP-1 cells co-incubated with CXCL13 (M13s.). ( I ) Western blot analyses of cytoplasmic and nucleic protein fractions of A549 cells transfected with control vector (V) or SPP1, or treated with Mϕs. or M13s. ( J ) Real-time PCR assays of indicated genes in A549 cells transfected with control vector or SPP1, or treated with Mϕs. or M13s. ( K ) Western blot analyses of lysates of A549 cells transfected with control vector (V) or SPP1, or treated with Mϕs. or M13s, using indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.09419.018

Article Snippet: The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Mutagenesis, Immunofluorescence, Transfection, Plasmid Preparation, Incubation

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Novel biomarker candidates for OSCC identified using microarray analysis.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques: Biomarker Assay, Microarray, Binding Assay

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Expression of CXCL13 mRNA in primary OSCC tissues by RT‐qPCR. (A) CXCL13 expression was higher in tumor tissues compared to adjacent normal tissues in all cases. (B) Significant upregulation of CXCL13 mRNA was observed in tumor tissues. ** p < 0.01 compared to adjacent normal tissues.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum levels of CXCL13 protein in patients with OSCC. (A) The average serum CXCL13 protein in 125 patients with OSCC was 72.8 ρg/mL. (B) The average serum CXCL13 protein in healthy individuals was 33.5 ρg/mL. (C) The reference value determined by the ROC curve for maximizing the TPF/FPF ratio was 53.0 ρg/mL. FPF, false‐positive fraction; TPF, true‐positive fraction.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 levels in patients with OSCC.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Diagnostic sensitivity of serum CXCL13 and SCC in patients with OSCC.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques: Diagnostic Assay

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 and clinicopathological factors in patients with OSCC. (A) Serum CXCL13 protein levels in T classification. The serum CXCL13 proteins increased in accordance with primary tumor size. The serum CXCL13 levels in T1 were significantly higher than in T3 and T4. (B) The serum CXCL13 proteins in recurrence. The serum levels of CXCL13 proteins in patients with recurrence were significantly elevated. (C) Patients with recurrence were classified into primary recurrence, neck LNM, and distant metastasis (including multiple responses). The serum levels of CXCL13 proteins in neck LNM were significantly elevated. * p < 0.05, ** p < 0.01.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques:

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Serum CXCL13 and prognosis in patients with OSCC. A comparison of overall survival (OS) and disease‐free survival (DFS) in patients with OSCC was made among the two groups, which were classified based on the median serum CXCL13 level using the Kaplan–Meier method with a log‐rank test. High expression of serum CXCL13 indicated poor prognosis in both OS (A, p = 0.044) and DFS (B, p = 0.018) across all stages. In Stage I/II, the groups had no significant difference in OS (C, p = 0.696) and DFS (D, p = 0.435). In Stage III/IV, both OS (E, p = 0.020) and DFS (F, p = 0.009) were significantly poorer in patients with high CXCL13 levels.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques: Comparison, Expressing

Journal: Cancer Medicine

Article Title: Serum CXCL13 as a Novel Biomarker in Oral Squamous Cell Carcinoma

doi: 10.1002/cam4.70263

Figure Lengend Snippet: Visium spatial transcriptome analysis using two cases of primary OSCC tissues. (A) Case 1: Primary tongue SCC tissues were classified into eight clusters based on the gene expression profile. Among these, the tumor area comprised four clusters (Cluster 1, 3, 5, and 6). CXCL13 expression was upregulated in three of four clusters within the tumor area. CD8 expression was detected in all tumor clusters. (B) Case 2: In Case 2, primary tongue SCC tissues were classified into seven clusters. The tumor area consisted of four clusters (Clusters 1, 2, 3, and 6). The expression of CXCL13 and CD8 was upregulated in three of four clusters within the tumor area. CD4 expression was detected only in Cluster 2 in the tumor area but not in Case 1.

Article Snippet: Serum CXCL13 protein levels were assessed with the use of the Human CXCL13/BLC/BCA‐1 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques: Expressing

Journal: Communications Biology

Article Title: Immunofibroblasts regulate LTα3 expression in tertiary lymphoid structures in a pathway dependent on ICOS/ICOSL interaction

doi: 10.1038/s42003-022-03344-6

Figure Lengend Snippet: a qPCR analysis of ltα mRNA transcripts from wt (black bars) salivary glands at day 0, 3 h, 6 h, day 1, day 2, day 5, day 8 and day 15 p.c. Gene expression was normalized to housekeeping gene β-actin and expressed as RQ values relative to day 0 mRNA transcripts results. b CD45+ cells expressing IL13, IL22 or LTα3 at day 2 p.c. (grey bars) and day 5 p.c. (white bars) in wt mice. c Enumeration of LTα + lymphocytes within the CD45+ cells in wt salivary glands at day 5 p.c. by flow cytometry. Representative dot plot of LTα expression in CD45+ cells and its frequency within CD3ε+ and B220+ cells. d – f Graphs showing qPCR analysis of ltα ( d ), ccl19 ( e ) and cxcl13 ( f ) mRNA transcripts from wt (black bars) and Cd3ε −/− (dark grey bars) salivary glands at day 5, 8 and day 15 p.c. g Quantification of ccl19 and cxcl13 mRNA transcripts from wt (black bars) and Ltα −/− (light grey bars) salivary glands at day 5, 8 and 15 p.c. In all cases, gene expression was normalized to housekeeping gene β-actin and expressed as RQ values relative to day 0 mRNA transcripts results. h Immunofluorescence staining in salivary glands from wt and Ltα −/− mice for lymphoid aggregates with CD3ε (red), CD19 (blue) and CXCL13 or CCL21 (green) at day 15 p.c. Scale bar 100 µm. i qPCR analysis of ccl19 and cxcl13 mRNA transcripts from wt (black bars) and Tnfr1/2 −/− (white bars) salivary glands at day, 5, 8 and 15 p.c. Chemokine mRNA transcript results were normalized to β-actin. j Immunofluorescence staining for lymphoid aggregates within infected salivary glands from wt and Tnfr1/2 −/− mice with CD3ε (red) and CD19 (blue) at day 8 p.c. Scale bar 100 µm. Data are mean ± s.e.m from two independent experiments with three to six mice analyzed per group. * p < 0.05; ** p < 0.01; *** p < 0.001, one-way ANOVA with Dunnett’s multiple comparison test ( a ) and unpaired t test ( b , d , e , f , g , i ).

Article Snippet: Quantitative real-time PCR was performed as previously described in , using following primers Ccl19 (Mm00839967_g1), Cxcl13 (Mm00444533_m1), Lta (Mm00440229_g1), Ltb (Mm00434774_g1), Tnf (Mm00443258_m1), Tnfrsf1a alias Tnfra (Mm00441883_g1), Icos (Mm00497600_m1), Icosl (Mm00497237_m1) and Actb alias β-actin (Mm01205647_g1) was used as an endogenous control.

Techniques: Gene Expression, Expressing, Flow Cytometry, Immunofluorescence, Staining, Infection, Comparison

Journal: Communications Biology

Article Title: Immunofibroblasts regulate LTα3 expression in tertiary lymphoid structures in a pathway dependent on ICOS/ICOSL interaction

doi: 10.1038/s42003-022-03344-6

Figure Lengend Snippet: a Representative dot plots showing flow cytometry staining for ICOS expression in wt salivary glands at day 5 p.c. by CD45+ cells. Graph showing percentage of ICOS+ T lymphocytes and NK cells infiltrating salivary glands at day 5 p.c. b viSNE plots of ICOS+ T lymphocytes from infected salivary gland from wt mice at day 5 p.c., analyzed by multicolor flow cytometry. Colours indicate cell expression level of labelled markers (LTα3 and CD4). c , d Flow cytometry analysis of absolute numbers of T cells and LTα-producing T cells in wt (black bars) and Icosl −/− (white bars) mice at day 5 p.c. e , f qPCR analysis of ltα and icos mRNA transcripts in FACS sorted CD3+ cells from wt (black bars) and Icosl −/− (white bars) mice at day 5 p.c. mRNA transcripts were normalized to housekeeping gene β-actin and presented as RQ values calculated with calibrator day 0 CD45+ cells. g wt mice were left untreated or treated with anti-ICOS blocking antibody from day 0 post salivary gland cannulation and sacrificed at days 5 and 8 p.c. Cannulated Icosl −/− were also sacrificed and analyzed at days 5 and 8 p.c. Representative microphotographs of salivary glands examined by immunofluorescence for CD3ε (red) and CD19 (blue) showing lymphocytic infiltration. Scale bar 100 µm. h qPCR analysis of mRNA obtained from salivary glands at day 5 and 8 p.c. of lymphoid chemokine ccl19 and cxcl13 in anti-ICOS treated wt mice or Icosl −/− mice (both white bars) in comparison to wt mice (black bars). mRNA transcripts were normalized to β-actin mRNA and results are presented as relative quantitation (RQ). i Transcript levels for ccl19, ccl21c and cxcl13 in FACS sorted CD45-EpCAM-CD31-pdpn+ immunofibroblasts from wt (black bars) and Icosl −/− (white bars) mice at day 5 p.c. Transcripts levels were normalised to β-actin. Data are mean ± s.e.m from two independent experiments with three to six mice analyzed per group. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. non-significant, unpaired t test.

Article Snippet: Quantitative real-time PCR was performed as previously described in , using following primers Ccl19 (Mm00839967_g1), Cxcl13 (Mm00444533_m1), Lta (Mm00440229_g1), Ltb (Mm00434774_g1), Tnf (Mm00443258_m1), Tnfrsf1a alias Tnfra (Mm00441883_g1), Icos (Mm00497600_m1), Icosl (Mm00497237_m1) and Actb alias β-actin (Mm01205647_g1) was used as an endogenous control.

Techniques: Flow Cytometry, Staining, Expressing, Infection, Blocking Assay, Immunofluorescence, Comparison, Quantitation Assay