Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: Homocysteine modulates CXCL10/CXCR3 axis activity to induce endothelial dysfunction.

doi: 10.14715/cmb/2024.70.2.28

Figure Lengend Snippet: Fig. 3. (A, B) qRT-PCR and Western blot analysis of CXCL10 and CXCR3 expression in HAECs treated with different concentrations of Hcy. (C) IHC analysis of the aorta in HHcy mice showing CXCL10 and CXCR3 expression. (D) qRT-PCR analysis of CXCL10 and CXCR3 expression in arterial endothelial cells.

Article Snippet: Additionally, HAECs were exposed to specific agents including Anti-CXCL10 antibodies (701225, Invitrogen, USA), Anti-CXCR3 antibodies (ab71864, Abcam, UK), IgG control antibodies (31154, Thermo Fisher, USA), NBI-74330 (a CXCR3 inhibitor, 4528, Tocris Bioscience, UK), and CXCL10 agonist (266-IP, R&D Systems, USA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: Journal of Neuro-Oncology

Article Title: Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

doi: 10.1007/s11060-024-04672-9

Figure Lengend Snippet: Cytokine expression and increased AKT activity are independent mechanisms without a tumor cell intrinsic effect on rebound growth. ( a ) Luminex-based multiplex assay results showing cytokine secretion during withdrawal after treatment for five days with either DMSO (solvent control) or 5 nM dabrafenib. Data is shown as mean ± SD ( n = 3 independent biological replicates). Two-tailed unpaired t-test, *p-value ≤ 0.05 **p-value ≤ 0.01; no indication: not significant. ( b ) Kinase phosphorylation array of samples treated for five days with 5 nM dabrafenib or DMSO (solvent control) followed by 24 h withdrawal (wd). Arrays consist of two membranes, each target is detected in technical duplicates. Images shown are representatives of two biological replicates. ( c - d ) Western blot analysis of AKT phosphorylation after five days treatment with 5 nM dabrafenib (dabra) followed by up to 72 h withdrawal. Blots shown are representative of three independent biological replicates ( c ). Quantification ( d ) is relative to solvent control (DMSO; dashed line) and shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test, * p-value ≤ 0.05; no indication: not significant. ( e - f ) Western blot analysis of AKT activity markers after treatment with recombinant cytokines. Treatment for five days with DMSO or 5 nM dabrafenib (dabra) served as negative and positive control for western blots ( e ). Blots shown are representative of three biological replicates ( e ). Quantification ( f ) was done relative to untreated samples (dashed line) and is shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test; no indication: not significant. ( g ) RT-qPCR analysis of chemokine gene expression after five days treatment with 5 nM dabrafenib (dabra) alone or in combination with varying concentrations of alpelisib (alp). Quantification was done relative to dabrafenib only treatment (0; dashed line) and is shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test, *p-value ≤ 0.05 **p-value ≤ 0.01; no indication: not significant. ( h ) Viable cell counts during treatment with 5nM dabrafenib (dabra) alone or in combination with a combination of antibodies neutralizing CCL2 (0.5 µg/mL), CX3CL1 (0.25 µg/mL), CXCL10 (0.25 µg/mL) and CCL7 (0.1 ng/mL) (neuABs), IgG control (1 µg/mL), 5 µM alpelisib (alp) or 1 µM ipatasertib (ipa) followed by ten days of withdrawal. Dashed line indicates withdrawal timepoint. Viable cell counts are normalized to treatment start (-5d). Data is shown on a logarithmic scale (base 10) as mean ± SD of at least three biological replicates. ( i ) Viable cell counts during treatment with 5 nM dabrafenib (dabra) followed by withdrawal. During withdrawal cells were either untreated (= solvent) or treated for five days with a combination of antibodies neutralizing CCL2 (0.5 µg/mL), CX3CL1 (0.25 µg/mL), CXCL10 (0.25 µg/mL) and CCL7 (0.1 ng/mL) (neuABs), IgG control (1 µg/mL), 5 µM alpelisib (alp) or 1 µM ipatasertib (ipa), followed by five days of withdrawal. Dashed lines indicate withdrawal timepoints. Viable cell counts are normalized to treatment start (-5d). Data is shown on a logarithmic scale (base 10) as mean ± SD of at least three biological replicates

Article Snippet: For cell count experiments and western blot analysis, cells were treated with antibodies neutralizing CCL2 (0.5 μg/ml; cat. no. MAB279, R&D systems), CX3CL1 (0.25 μg/ml; cat. no. MAB3652, R&D systems), CXCL10 (0.25 μg/mL; cat. no. MAB266, R&D systems) and CCL7 (0.1ng/ml; cat. no. MAB282, R&D systems) or mouse IgG (cat. no. MAB002, R&D systems).

Techniques: Expressing, Activity Assay, Luminex, Multiplex Assay, Solvent, Control, Two Tailed Test, Phospho-proteomics, Western Blot, Recombinant, Positive Control, Quantitative RT-PCR, Gene Expression

Journal: Journal of Neuro-Oncology

Article Title: Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

doi: 10.1007/s11060-024-04672-9

Figure Lengend Snippet: Increased cytokine secretion upon MAPKi treatment and withdrawal induces increased microglia attraction. ( a ) Schematic of transwell migration assay setup to investigate migration of HMC3 cells towards conditioned media (CM) collected from BT-40. ( b ) Transwell migration assay of HMC3 cells towards conditioned media (CM) collected from BT-40 cells after 24 h of withdrawal after five days treatment with DMSO (solvent control), 5nM dabrafenib (d) or 2.7 nM dabrafenib and 0.3 nM trametinib (d + t). 2% FCS serves as baseline control as CM contains 2% FCS, 0% FCS as negative control and 10% FCS as positive control. Quantification is shown as mean ± SD ( n = 3 independent biological replicates; 2 technical duplicates per condition; 10–12 randomly distributed images were quantified per transwell) relative to 2% FCS. One-sample t-test, * p-value ≤ 0.05; no indication: not significant. ( c ) Transwell migration assay of HMC3 cells towards CM collected 24 h after dabrafenib withdrawal (dabra wd) containing either antibodies neutralizing CCL2 (1 µg/mL), CX3CL1 (0.5 µg/mL), CXCL10 (0.5 µg/mL) and CCL7 (0.2 ng/mL) (neuABs) or IgG (2 µg/mL). 2% FCS serves as baseline control as CM contains 2% FCS, 0% FCS as negative control and 10% FCS as positive control. Quantification is shown as mean ± SD ( n = 3 independent biological replicates; 2 technical duplicates per condition; 10–12 randomly distributed images were quantified per transwell) relative to 2% FCS. One-sample t-test and two-tailed unpaired t-test (comparing IgG to neuABs), *p-value ≤ 0.05 ***p-value ≤ 0.001; no indication: not significant. ( d ) Representative fluorescence images showing HMC3 migrated through the transwell, nuclei were stained with DAPI. Scale bar = 50 µM. ( e - h ) RT-qPCR analysis of CX3CL1 ( e ), CXCL10 ( f ), CCL2 ( g ) and CCL7 ( h ) expression in BT-40 xenograft tumors after six days treatment with dabrafenib (dabra; 100 mg/kg, six doses, once daily) followed by three days of withdrawal (withdrawal). Samples from mice showing tumor progression (#4, #6; Fig. d) during dabrafenib treatment were excluded from the analysis. Quantification was done relative to the median of untreated samples (control). CX3CL1 was undetected in one sample (#2), CCL2 was undetected in two samples (#2, #7) and CCL7 was undetected in six samples (#2, #3, #5, #7, #8, #11), for these samples Ct values were set to 40 (max. number of cycles). Boxplots depict the median, first and third quartiles. Whiskers extend from the hinge to the largest/smallest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range). Two-tailed unpaired t-test; no indication: not significant (control: n = 3 mice, dabra: n = 4 mice, withdrawal: n = 6 mice)

Article Snippet: For cell count experiments and western blot analysis, cells were treated with antibodies neutralizing CCL2 (0.5 μg/ml; cat. no. MAB279, R&D systems), CX3CL1 (0.25 μg/ml; cat. no. MAB3652, R&D systems), CXCL10 (0.25 μg/mL; cat. no. MAB266, R&D systems) and CCL7 (0.1ng/ml; cat. no. MAB282, R&D systems) or mouse IgG (cat. no. MAB002, R&D systems).

Techniques: Transwell Migration Assay, Migration, Solvent, Control, Negative Control, Positive Control, Two Tailed Test, Fluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 1. Circulating CXCL10 in leprosy patients and healthy con- trols. Each point represents one sample from a different individual. The groups were healthy controls, borderline tuberculoid without re- action (BT), borderline lepromatous without reaction (BL), polar lep- romatous without reaction (LL), BT with reaction (BT plus RR), BL with reaction (BL plus RR), and LL patients with T2R (erythema nodosum leprosum). For patients with reaction (BT plus RR, BL plus RR, and T2R), the sample was taken at the time of reaction, prior to treatment. For LL, n 6; for all other groups, n 10. The horizontal lines indicate median values. The median level of CXCL10 in LL patients with type 2 reaction was not significantly elevated (P 0.9).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques:

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 2. Circulating CXCL10 in repeated samples from leprosy patients who developed T1R. Each graph depicts the measurements made from serum samples obtained at 1 month intervals in borderline tuberculoid (A, no. 1 to 10) and borderline lepromatous (B, no. 11 to 20) patients. All patients with clinical T1R received standard multidrug treatment starting at the first visit. The arrows indicate the visits at which T1R was diagnosed. The number above each time point indicates the dose of prednisone started on that day, tapered monthly as indicated; in some cases, corticosteroids were not used initially but were started 1 to 2 months after the initial clinical diagnosis of T1R. Each serum specimen was obtained before corticosteroid treatment was initiated. CXCL10 was significantly elevated during T1R in the 10 BT patients (A) (P 0.0006) and the 10 BL patients (B) (P 0.0001). In patients 12, 16, and 17 (B), T1R was diagnosed by histopathological criteria only; when they were excluded from the analysis, the association of T1R with CXCL10 remained significant (P 0.05).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Biomarker Discovery

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 3. Expression levels of CXCL10 and IFN- genes in skin biopsy specimens from leprosy patients. (A to H) Real-time quantitative RT-PCR was performed on cDNA obtained from sequential skin biopsy specimens from individual leprosy patients who were placed on MDT, none of whom had T1R at the time of the first biopsy. Five patients (A to E) had clinical symptoms of T1R at the time of the second biopsy; two of these (C and D) had a third biopsy after the reaction had resolved. Three patients (F to H) had no clinical symptoms of T1R at the time of the initial or second biopsy. Data were obtained using the standard-curve method and were normalized using 18S rRNA values, repeated three times for all determinations. The results are presented as the mean standard deviation.

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

Journal: medRxiv

Article Title: Magnitude and timing of the antiviral response determine SARS-CoV-2 replication early in infection

doi: 10.1101/2021.01.22.21249812

Figure Lengend Snippet: (A) Volcano plot showing significantly differentially expressed protein-coding genes based on RNASeq of NP swab RNA from SARS-CoV-2 patients (n=30) compared to control SARS-CoV-2 negative subjects (n=8). Transcripts with fold change>2, adjusted p-value<0.05 are highlighted in red. (B) Top 20 ingenuity pathways enriched in SARS-CoV-2+ compared to controls, based on 1770 differentially expressed RNAs. P-value and Z-score for each pathway is indicated on the x-axis. Pathways related to interferon and interferon regulatory factor (IRF) signaling are highlighted in red. (C) Transcription factor binding sites associated with NP transcripts enriched in SARS-CoV-2+ patients compared to controls. Bars show strength of association of motifs/tracks with enriched transcripts, indicated by NES score. Y-axis label indicates top transcription factor associated with each cluster of motifs (M) or tracks (T) and the cluster code. Number of enriched transcripts associated with each track/motif is indicated to the right of each bar. Transcription factors associated with the interferon response are highlighted in red. (D) Graphical summary of pathways and regulators enriched based on ingenuity pathway analysis of differentially expressed genes enriched in NP RNA of SARS-CoV-2+ patients compared to controls. (E) Heatmap showing relative expression level of top 45 most significant differentially expressed genes in patients (left) or SARS-CoV-2-negative controls (right). Clinical characteristics of each patient are indicated by color: viral load (red=highest viral load/lowest Ct value, green=lowest viral load/highest Ct value); NP CXCL10 protein level (red=highest, green=lowest, white=data not available). Heatmap colors represent values from highest (red) to lowest (green) for viral load (based on Ct value), CXCL10 concentration (pg/ml), or gene expression level, scaled from minimum to maximum (green=0; yellow=0.5, red=1) Patient characteristics indicated at the top of the graph include Admission status (grey=outpatient, black= admitted); Gender (blue=male, pink=female); Age (blue<55yrs, purple>60 yrs.) White = data not available. (F) Correlation between reads mapping to CXCL10 and reads mapping to other ISGs (Ifit2, OasL, Isg15). (G) Correlation between reads mapping to Cxcl10 and CXCL10 protein measured by ELISA in NP swab-associated viral transport medium.

Article Snippet: CXCL10 levels in cell-free residual nasopharyngeal swab samples or tissue culture supernatants was quantified using a solid phase sandwich enzyme linked immunosorbent assay (ELISA) (Cat no: DY266, R&D systems, MN).

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

Journal: medRxiv

Article Title: Magnitude and timing of the antiviral response determine SARS-CoV-2 replication early in infection

doi: 10.1101/2021.01.22.21249812

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CXCL10 levels in cell-free residual nasopharyngeal swab samples or tissue culture supernatants was quantified using a solid phase sandwich enzyme linked immunosorbent assay (ELISA) (Cat no: DY266, R&D systems, MN).

Techniques: Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins induce CXCL10 in a cell type-specific manner. ( A ) SDS-polyacrylamide gel electrophoresis of recombinant human galectins (rhGal) stained with Coomassie Blue. Molecular weights of protein standards are indicated on the left. The hemagglutination assay was performed using serial two-fold dilutions of the recombinant protein. The bottom row contains protein incubated with β-lactose, a competitive carbohydrate inhibitor of galectin binding. ( B ) Cultures of human corneal fibroblasts, human corneal epithelial cells, THP-1 monocytes and human umbilical vein endothelial cells (HUVECs) were incubated with 50 µg/mL rhGal-1, -3 and -8 or 50 µg/mL IFN-γ. Quantitative real-time PCR was used to determine the levels of CXCL10 mRNA after 6 h of incubation ( n = 3 independent experiments), whereas ELISA was used to determine the levels of CXCL10 protein in cell culture supernatants after 24 h of incubation ( n = 6 independent experiments). The data represent the mean ± SEM.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Polyacrylamide Gel Electrophoresis, Recombinant, Staining, Hemagglutination Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins differentially dictate fibroblast-dependent immune cell chemotaxis. ( A ) Recombinant human galectin (rhGal)-1, -3 and -8 were incubated with fibroblasts for 24 h. The conditioned media were pre-cleared with β-lactose Sepharose beads three times to remove galectins prior to chemotaxis assays. The presence of galectins in pre-cleared conditioned media was evaluated by immunoblotting. ( B ) Quantification by flow cytometry of the migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with increasing concentrations of recombinant galectins or BSA ( n = 3 independent experiments performed in duplicate). ( C ) Migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with 50 μg/mL of recombinant galectins or BSA. A CXCL10 neutralizing antibody was added to the conditioned media in neutralization studies ( n = 4–5 independent experiments in duplicate). The data in ( B ) represent the mean ± SEM. The box and whisker plots show the 25 and 75 percentiles (box), the median, and the minimum and maximum data values (whiskers). Significance was determined using Student’s t test. ** p < 0.01.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Chemotaxis Assay, Recombinant, Incubation, Western Blot, Flow Cytometry, Migration, Labeling, Neutralization, Whisker Assay