Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Overview of all differentially expressed genes categorized according to their activity in cancer biology.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Validation of differential gene expression in MCC cell lines on protein level and mRNA level. (A) TPM values were acquired from our sequencing output. The data represent mean values ± SEM from three independent experiments (two for MKL-1 control). (B) After 72 h of treatment with or without IFNγ (3,000 U/ml), the Merkel cell carcinoma cell lines MKL-1, MKL-2, and WaGa were stained for signal transducer and activator of transcription 1-alpha/beta (STAT1), bone marrow stromal antigen 2 (BST2), C-X-C motif chemokine 10 (CXCL10), and HLA class II histocompatibility antigen gamma chain (CD74) and analyzed by flow cytometry. The average MFI of three independent experiments ± standard error is indicated. p -values were determined using the paired student’s t -test. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Biomarker Discovery, Gene Expression, Sequencing, Control, Staining, Flow Cytometry

Journal: Cancers

Article Title: EGFR-Tyrosine Kinase Inhibitors Induced Activation of the Autocrine CXCL10/CXCR3 Pathway through Crosstalk between the Tumor and the Microenvironment in EGFR-Mutant Lung Cancer.

doi: 10.3390/cancers15010124

Figure Lengend Snippet: Figure 2. Cytokine array analysis of coculture supernatants during EGFR-TKI treatment. EGFR-mutant, EGFR-TKI (erlotinib)-sensitive cell lines (HCC 4006 and HCC827), EGFR wild-type, EGFR-TKI resistant cell line (A549), and EGFR-mutant, EGFR-TKI (erlotinib)-resistant cell line (H1975) indirectly cocultured with activated PBMC with or without erlotinib (10 nM for HCC827 and 40 nM for HCC4006, H1975, and A549) for 48 h. (A,B) CXCL10 (dot #4) was elevated after coculture, whereas erlotinib treatment had no effect on CXCL10 expression levels in HCC4006, HCC827, and H1975 cells. Spot densities from each membrane are described in Supplementary Table S1. (C,D) ELISA analysis of supernatants cocultured with activated PBMCs and two EGFR-mutant, EGFR-TKI-sensitive cell lines (HCC4006 and HCC827, respectively) during erlotinib treatment©. Data are presented as mean ± SD of three independent experiments. Statistical significance was determined with an unpaired Student’s t-test (* p < 0.05, ** p < 0.01 versus cultured HCC4006 cells or HCC827 cells alone, respectively).

Article Snippet: For immunohistochemistry analysis of CXCL10, HIF-1α, and p-Src, deparaffinized tissue sections were stained with antibodies to CXCL10 (1:200, St John’s Laboratory Ltd., London, UK), HIF-1α (1:200, Santa Cruz Biotechnology), and p-Src (1:500, Cell Signaling Technology) at 4 ◦C overnight.

Techniques: Mutagenesis, Expressing, Membrane, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancers

Article Title: EGFR-Tyrosine Kinase Inhibitors Induced Activation of the Autocrine CXCL10/CXCR3 Pathway through Crosstalk between the Tumor and the Microenvironment in EGFR-Mutant Lung Cancer.

doi: 10.3390/cancers15010124

Figure Lengend Snippet: Figure 3. Activation of the oncogenic CXCR3-Src pathway in EGFR-mutant lung cancer cells by CXCL10. The expression of p-Src, p-STAT3, p-p65, and HIF-1α from HCC4006 whole cell lysates was detected with Western blotting. To explore the oncogenic CXCR3 pathway, we pretreated HCC4006 cells with 0.01–1 µg/mL anti-CXCR3 antibody for 2 h, 5 nM osimertinib for 24 h, and 50 nM erlotinib for 24 h, then administered treatment with 25 ng/mL CXCL10 for 30 min (A) or 24 h (B). (A) CXCL10 stimulated p-Src expression, and anti-CXCR3 antibody decreased p-Src expression, in a dose-dependent manner. Neither osimertinib nor erlotinib affected the expression of p-Src. (B,C) p- STAT3, p-p65, and HIF-1α, analyzed by Western blotting (B) and immunofluorescence. Increased localization of p-p65 (green) and HIF- 1α (red) in the nuclei was observed (C).

Article Snippet: For immunohistochemistry analysis of CXCL10, HIF-1α, and p-Src, deparaffinized tissue sections were stained with antibodies to CXCL10 (1:200, St John’s Laboratory Ltd., London, UK), HIF-1α (1:200, Santa Cruz Biotechnology), and p-Src (1:500, Cell Signaling Technology) at 4 ◦C overnight.

Techniques: Activation Assay, Mutagenesis, Expressing, Western Blot

Journal: Cancers

Article Title: EGFR-Tyrosine Kinase Inhibitors Induced Activation of the Autocrine CXCL10/CXCR3 Pathway through Crosstalk between the Tumor and the Microenvironment in EGFR-Mutant Lung Cancer.

doi: 10.3390/cancers15010124

Figure Lengend Snippet: Figure 4. Autocrine activation of the CXL10/CXCR3 pathway by endogenous CXCL10 in EGFR-mutant lung cancer cells. (A) Supernatants collected from HCC4006 and HCC827 cells treated with 20 pg/mL IFN−γ for 72 h were analyzed using ELISA for CXCL10 (* p < 0.5 and ** p < 0.01 versus control). (B) Increased CXCL10 mRNA expression of HCC 4006 with coculture and treatment of EGFR-TKI. (C–E) HCC4006 and HCC 827 cells cocultured with activated PBMCs for 48 h and treated for 6 h with siRNA against endogenous CXCL10. (C) ELISA quantification for CXCL10 of supernatants from coculture with activated PBMC (* p < 0.05). (D) Western blot analysis of samples treated with siRNA against CXCL10. € Immunofluorescence analysis of p-p65 and HIF-1α in cells treated with siRNA CXCL10 or erlotinib (50 nM, HCC4006 cell and 20 nM HCC827 cells). Nuclei were counterstained with DAPI (blue) for the detection of DNA. Data are presented as mean ± SD of three independent experiments. Cells were treated with 100 µM of each of the STAT3 and HIF-1α inhibitors.

Article Snippet: For immunohistochemistry analysis of CXCL10, HIF-1α, and p-Src, deparaffinized tissue sections were stained with antibodies to CXCL10 (1:200, St John’s Laboratory Ltd., London, UK), HIF-1α (1:200, Santa Cruz Biotechnology), and p-Src (1:500, Cell Signaling Technology) at 4 ◦C overnight.

Techniques: Activation Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Control, Expressing, Western Blot, Immunofluorescence

Journal: Cancers

Article Title: EGFR-Tyrosine Kinase Inhibitors Induced Activation of the Autocrine CXCL10/CXCR3 Pathway through Crosstalk between the Tumor and the Microenvironment in EGFR-Mutant Lung Cancer.

doi: 10.3390/cancers15010124

Figure Lengend Snippet: Figure 5. Effects of CXCL10/CXCR3–Src pathway activation by TKIs in a transgenic mouse model of EGFR-mutant lung cancer. TetO–EGFRL858R transgenic mice were fed 200 mg/kg of doxycycline– impregnated feed. After 12–14 weeks, tumor development was confirmed with an MRI scan, and mice were orally administered erlotinib (12.5 mg/kg) daily together with doxycycline. (A) BAL fluid from the lungs of transgenic mice was used to collect CXCL10 on day 1 (n = 5, 3 male and 2 female), day 3 (n = 7, 5 male and 2 female), day 7 (n = 7, 5 male and 2 female), and no treatment (n = 5, 3 male and 2 female). Data are presented as mean ± SD. Statistical significance was determined with Mann–Whitney U test (* p < 0.05 and ** p < 0.01). (B) Proportion of CXCL10 expressed tumor cells among viable persistent tumor cells according to erlotinib treatment on day 1 (n = 5, 3 male and 2 female), day 3 (n = 7, 5 male and 2 female), day 7 (n = 7, 5 male and 2 female), and no treatment (n = 5, 3 male and 2 female). Data are presented as mean ± SD. Statistical significance was determined with Mann–Whitney U test (* p < 0.05). (C,D) Levels of CXCL10, HIF-1α, and p-Src expression in persistent lung tumor sections after erlotinib treatment as indicate€ (E) Representative immunohistochemical staining of p-Src in persistent lung tumors extracted from mice carrying the EGFR-L858R mutation and treated with erlotinib on day 7. (F) Positive correlation in overlapping clusters between p-Src and HIF-1α and EGFR-TKI response. Pearson’s correlation analysis (r = 0.72, p = 0.0018) (doxycycline (n = 3), day 1 (n = 6), day 3 (n = 4), day 7 (n = 5)).

Article Snippet: For immunohistochemistry analysis of CXCL10, HIF-1α, and p-Src, deparaffinized tissue sections were stained with antibodies to CXCL10 (1:200, St John’s Laboratory Ltd., London, UK), HIF-1α (1:200, Santa Cruz Biotechnology), and p-Src (1:500, Cell Signaling Technology) at 4 ◦C overnight.

Techniques: Activation Assay, Transgenic Assay, Mutagenesis, MANN-WHITNEY, Expressing, Immunohistochemical staining, Staining

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: ( A ) Top 10 terms in GO analysis ( P <0.05). ( B ) Enriched terms in KEGG pathway analysis ( P <0.05). ( C ) Top three PPI networks of CXCL10, KRT, and ZAP70 modules. The network includes three modules and 14 nodes showing the association between tumor microenvironment-related genes and PTC. ( D ) Bar chart signifying important genes (degree>10) in the PPI network.

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: ( A ) Correlations between CXCL10 and differentially expressed genes in PTC. ( B and C ) Heatmaps indicate the top 50 genes positively and negatively correlated with CXCL10 in PTC. Red refers to positively correlated genes and green refers to negatively correlated genes. Statistical analysis was performed using Pearson’s test. GO function ( D ) and KEGG pathway ( E ) analyses of CXCL10.

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: Diseases related to CXCL10 (Open Targets)

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of CXCL10 gene expression

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques: Gene Expression

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: The kinase target networks of CXCL10 in PTC (LinkedOmics)

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: PPI networks of CXCL10 and related genes (GeneMANIA)

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: (CXCL10 protein levels were higher in tumor-adjacent tissues compared to PTC tissues; 400× magnification).

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: Relationship between CXCL10 and various infiltrating immune cells (TIMER)

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: Correlation analysis between CXCL10 and gene biomarkers of immune cells in THCA (TIMER)

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal: Bioscience Reports

Article Title: CXCL10 is a potential biomarker and associated with immune infiltration in human papillary thyroid cancer

doi: 10.1042/BSR20203459

Figure Lengend Snippet: Drug resistance analysis of the central gene, CXCL10, using GSCALite

Article Snippet: After overnight incubation with the rabbit monoclonal anti-CXCL10 antibody (1:100 dilution, BOSTER, Wu Han, China, BA4723) at 4°C, 120-point PTC tissue microarrays were washed with PBS and incubated with broad-spectrum secondary antibodies.

Techniques:

Journal:

Article Title: Stem cells as potential therapeutic targets for inflammatory bowel disease

doi:

Figure Lengend Snippet: New therapies in development or clinical evaluation for the treatment of IBD

Article Snippet: Anti-CXCL10 antibody , Protein Design Labs , CD.

Techniques: Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: Ready-to-use iPSC-derived microglia progenitors for the treatment of CNS disease in mouse models of neuropathic mucopolysaccharidoses

doi: 10.1038/s41467-024-52400-8

Figure Lengend Snippet: A Schematic of the differentiation process for MG01. B Total MG01 cells produced in a T-75 flask for 3 hiPSC lines. C Viability of MG01 after thaw for each of the 3 hiPSC lines. D Representative flow-cytometry plots for CD45/CD14 and CX3CR1/CD11B in post-thaw MG01 and graphs depicting quantification of 3 biological replicates per line. E Panel of representative images depicting immunoreactivity of MG01 (Line 82) to CD45, CD68, P2RY12, TREM2, IBA1, TMEM119 and PU.1 after two days in culture. F Representative phase contrast images of polarized MG01 (Line 82) 12 hours after the addition of pHrodo-conjugated E. coli particles. Upper left insets depict the detected fluorescence of pHrodo particles after phagocytosis. G Pro-inflammatory cytokine (TNFα, CXCL10 and IL-6) expression levels after polarization of MG01 to M1-like state using LPS and IFNγ. H Quantification of representative phagocytosis assay (2DIV). Graph depicts pHrodo fluorescence after addition to MG01. Images were acquired every 30 m for a span of 24 h. Data points in B , and C represent biological replicates; in D represent a combination of biological and technical replicates, in G – H represent technical replicates from one representative experiment. All cell lines tested behaved similarly and data depicted in E – H is representative of all hiPSC-derived MG01. Graphs denote mean +/− SEM. Scale bars in panel E represent 50 µm. Panel A was in part created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: Supernatants were thawed at room temperature and 16 µL were transferred to small-volume 96 well plates for the addition of donor and acceptor antibodies from either the IL-6, TNFα, or CXCL10 kit (Cisbio, 62HIL06PEG, 62HTNFAPEG, 62HCX10PEG).

Techniques: Produced, Flow Cytometry, Fluorescence, Expressing, Phagocytosis Assay, Derivative Assay