Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet:

Article Snippet: Mouse IL-10 ELISA kit , Biolegend , Cat#431414.

Techniques: Activation Assay, Recombinant, Avidin-Biotin Assay, Plasmid Preparation, Staining, Diagnostic Assay, Magnetic Beads, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Isolation, Purification, Expressing, Microarray

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet:

Article Snippet: Recombinant human FGFR3 , Sino Biological , 16486-H08H.

Techniques: Purification, Recombinant, Labeling, Virus, Microarray, Cytokine Assay, Transfection, Chromatin Immunoprecipitation, ChIP-sequencing, Plasmid Preparation, Software, Imaging

Journal: Nature Communications

Article Title: Tumour-associated macrophages exhibit anti-tumoural properties in Sonic Hedgehog medulloblastoma

doi: 10.1038/s41467-019-10458-9

Figure Lengend Snippet: CCL2 is a key monocyte chemo-attractant cytokine in SHH MB. a Schematic of the experiment and results of cytokine analysis. NeuroD2:SmoA1 ; Atoh1:GFP tumour GFP tissue was extracted under a fluorescence microscope and either dissociated and FACS-sorted to isolate GFP + cells or homogenised ( SmoA1 MB); naive cerebellum was used as a control (CB tissue). Images are representative of three independent experiments. b ELISA analysis of CCL2 cytokine level in cerebellum (CB) and medulloblastoma (MB) tissue, N = 9, Mann–Whitney U test, ** P < 0.01. c , d Relative Ccl2 ( c ) and Ccr2 ( d ) expression levels in FACS-sorted cells, GFP + are tumour cells, GFP − are cells of the tumour microenvironment. N = 6, Mann–Whitney U test, ** P < 0.01. e Microarray data analysis of human patient cohort for CCL2 gene . WNT Wingless, SHH Sonic Hedgehog, N = 140 for WNT, N = 437 for SHH, N = 144 for group 3, and N = 326 for group 4. Box plots represent median and quratiles with whiskers indicating range of values. One-way ANOVA with multiple comparisons, F = 19.17, **** P < 0.0001. Data are represented as mean ± S.E.M. for b – d

Article Snippet: Mouse Cytokine array panel A (ARY006, R&D systems) was used to measure cytokine levels in various cells and tissues using the manufacturer’s provided protocol.

Techniques: Fluorescence, Microscopy, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Expressing, Microarray

Journal: bioRxiv

Article Title: Oligodendrocyte Precursor Cells Are Co-Opted by the Immune System to Cross-Present Antigen and Mediate Cytotoxicity

doi: 10.1101/461434

Figure Lengend Snippet: Isolated and PDGF (20 ng/mL) expanded postnatal rat OPCs (P4-P6) were differentiated with T3 ( ), T3 + IFNγ (10 ng/mL; ), T3 + IL17 (50 ng/mL; ), or T3 + IFNγ + IL-17 ( ) for 96 hrs prior to assessment using qPCR (a) and ICC staining for MBP and Olig2 (b) . Isolated and PDGF (20 ng/mL) expanded postnatal rat OPCs (P4-P6) were left undifferentiated (0 hrs; ) or differentiated with T3 (10 nM), IFNγ (10 ng/mL), T3 + IFNγ, for 8 hrs ( ), 24 hrs ( ), 48 hrs ( ) and 96 hrs ( ) to compare gene expression between groups while obtaining information at multiple time points. (c-e) Affymetrix gene arrays analysis was performed from three independent biological replicates. (c) Venn diagram summarizing the number and overlap of up-regulated (top) and down-regulated (bottom) genes based on time point. (d) Global heat map of all probes clustered into three gene expression patterns comparing PDGF, T3, and T3 + IFNγ at all time points. (e) Targeted heat map comparing PDGF, T3, IFNγ, and T3 + IFNγ at all time points. Displayed genes were identified from GSEA analysis (supplement 3). (f) Quantitative PCR validation of OPCs differentiated with T3 ( ), T3 + IFNγ (10 ng/mL; ) or T3 + IL17 (50 ng/mL; ) for 96 hrs prior to assessment. Error bars represent the standard deviation from 3 independent primary isolations and experiments. Significance for qPCR analysis was determined by one-way ANOVA analysis followed by Dunnett’s multiple comparison analysis where T3 + IFNγ or T3 + IL17 were compared to T3 alone control (P * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001).

Article Snippet: To determine the cytokine effects on OPC differentiation, microarray and qPCR, rat recombinant IFNγ (10 ng/mL; Peprotech) or rat recombinant IL-17 (50 ng/mL; R&D Systems) were supplemented.

Techniques: Isolation, Staining, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Comparison

Journal: Frontiers in Immunology

Article Title: MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation

doi: 10.3389/fimmu.2022.953714

Figure Lengend Snippet: IL-17 is necessary to maintain asthmatic phenotype in HDM-induced murine model. ( A) The level of IL-17A in mouse Bronchoalveolar lavage fluid (BALF) detected by Bio-Plex. N=10. (B, C) IHC staining (B) and quantitative analysis (C) of IL-17 positive cells in mouse lung tissue section. The brown area represents IL-17 positive. Black arrows indicate stained macrophage. Scale bar =50μm. N=10. (D, E) IHC staining (D) and quantitative analysis (E) of airway smooth muscle mass (indicated by Alpha-Smooth Muscle Actin) in mouse lung tissue section. The brown areas represent the airway smooth muscle cells. Scale bars= 100μm. N=10. (F, G) collagen deposition analysis in mouse lung tissue. Collagen was stained by Picro Sirius red, the color red indicates the positive staining. Scale bars=100μm. N=10. *, P<0.05, **, P<0.01, ***, P<0.001. n.s., not statistically significant.

Article Snippet: The immunohistochemistry staining for IL-17A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-SMA (Santa Cruz Biotechnology) was performed on paraffin-embedded mouse lung tissue sections as previously described ( ).

Techniques: Immunohistochemistry, Staining

Journal: Frontiers in Immunology

Article Title: MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation

doi: 10.3389/fimmu.2022.953714

Figure Lengend Snippet: Indispensable role of IL-17 in the inflammatory response of HDM-induced asthma in murine model. (A) Counts of inflammatory cells in BALF. N=10. (B) The level of inflammatory cytokines in BALF. The inflammatory cytokines in BALF were detected by Bio-Plex. N=10. *, P<0.05, **,P<0.01, ***, P<0.001. n.s., not statistically significant.

Article Snippet: The immunohistochemistry staining for IL-17A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-SMA (Santa Cruz Biotechnology) was performed on paraffin-embedded mouse lung tissue sections as previously described ( ).

Techniques:

Journal: Frontiers in Immunology

Article Title: MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation

doi: 10.3389/fimmu.2022.953714

Figure Lengend Snippet: miR-365-3p is downregulated in wildtype HDM-induced mice but not in IL-17 KO HDM-induced mice. (A) Microarray of dysregulated miRNA in wildtype saline and HDM-induced mice, N=3. (B) The level of miR-365-3p examined by qRT-PCR in wild-type and IL-17KO mice. N=10. (C) The effect of IL-17 in miR-365-3p expression in MLE-12 cells. The MLE-12 cells were treated by IL-17, and the miR-365-3p expression was tested by qRT-PCR N=4. *, P<0.05, **, P<0.01, ***, P<0.001. n.s., not statistically significant.

Article Snippet: The immunohistochemistry staining for IL-17A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-SMA (Santa Cruz Biotechnology) was performed on paraffin-embedded mouse lung tissue sections as previously described ( ).

Techniques: Microarray, Quantitative RT-PCR, Expressing

Journal: Frontiers in Immunology

Article Title: MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation

doi: 10.3389/fimmu.2022.953714

Figure Lengend Snippet: ARRB2 is the target gene of miR-365-3p to negate proinflammatory effect of IL-17. (A) target genes of miR-365-3p that are related with asthma. (B) mRNA expression of A RRB2 detected by qRT-PCR. N=5. (C) The targeting role of miR-365-3p on A RRB2 examined by reporter fluorescence assays, N=3. (D) Effect of miR-365-3p rescue on mRNA level of A RRB2. N=6. (E) Effect of miR-365-3p rescue on protein level of ARRB2. (F) regulatory effect of miR-365-3p on ARRB2 expression in both mouse MLE-12 (left) and human A549 (right) epithelial cells. (G) immunoblotting analysis of ARRB2 overexpression from pcDNA3.1 vector in MLE-12 cells. (H) expression of KC and IL-6 in ARRB2-overexpression MLE-12 cells upon IL-17 treatment. N=4. (I) immunoblotting analysis of ARRB2 knockdown by siRNA in MLE-12 cells. (J) expression of KC and IL-6 in ARRB2-knockdown MLE-12 cells upon IL-17 treatment. N=4. *, P<0.05, **, P<0.01, ***, P<0.001. n.s., not statistically significant.

Article Snippet: The immunohistochemistry staining for IL-17A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-SMA (Santa Cruz Biotechnology) was performed on paraffin-embedded mouse lung tissue sections as previously described ( ).

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Western Blot, Over Expression, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation

doi: 10.3389/fimmu.2022.953714

Figure Lengend Snippet: miR-365-3p negates IL-17-induced inflammatory cytokines expression in murine epithelial cells and macrophages. (A) Effect of miR-365-3p mimic on mRNA expression of KC and IL-6 in MLE-12 cells upon IL-17 treatment. N=5. (B) Effect of miR-365-3p mimic on mRNA expression of IL-6 in J774a.1 cells upon IL-17 treatment. N=3. *, P<0.05, ***, P<0.001. n.s., not statistically significant.

Article Snippet: The immunohistochemistry staining for IL-17A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-SMA (Santa Cruz Biotechnology) was performed on paraffin-embedded mouse lung tissue sections as previously described ( ).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: MiR-365-3p is a negative regulator in IL-17-mediated asthmatic inflammation

doi: 10.3389/fimmu.2022.953714

Figure Lengend Snippet: The proposed scheme of negative regulating role of miR-365-3p in IL-17 induced KC and IL-6 production. miR-365-3p, which was diminished by IL-17 in murine and human asthmatic pathogenesis, functioned as an essential negative mediator to suppress IL8 (KC for mouse) and IL-6 upon IL-17 stimulation by targeting ARRB2.

Article Snippet: The immunohistochemistry staining for IL-17A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and α-SMA (Santa Cruz Biotechnology) was performed on paraffin-embedded mouse lung tissue sections as previously described ( ).

Techniques:

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: Protein microarray analysis of A mouse serum using R&D Systems Proteome Profiler Mouse XL Cytokine Array in the presence (rat IgG) and absence (α-Ly-6G) of Nϕ 24 h post-TBI and B Nϕ isolated from BM that were ex vivo treated with vehicle or entolimod for 30 min. Measurement by ELISA of MMP-9 in C serum from mice ( n = 5/group) in the presence (rat IgG) and absence (α-Ly-6G) of Nϕ 2 h post-treatment with vehicle or entolimod (TBI was delivered 24 h prior to vehicle or entolimod) and D supernatants from Tlr5 WT/WT and Tlr5 −/− Nϕ isolated from BM ( n = 6 mice/group) and ex vivo treated with vehicle or entolimod for 30 min.

Article Snippet: We purchased mouse MethoCult GF and MethoCult without cytokines from STEMCELL Technologies, recombinant mouse G-CSF from Peprotech, rat IgG2a, κ (clone 2A3) and α-Ly-6G (clone 1A8) from BioXcell, Proteome Profiler Mouse XL Cytokine Array from R&D Systems, Duoset ELISA kits from R&D Systems, and recombinant mouse MMP-9 from BioLegend.

Techniques: Microarray, Isolation, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Specific cannabinoids revive adaptive immunity by reversing immune evasion mechanisms in metastatic tumours

doi: 10.3389/fimmu.2022.982082

Figure Lengend Snippet: Phytocannabinoids, but not endocannabinoids, induce MHC-I expression in a dose- and time-dependent manner. (A) The endocannabinoids 2-AG and AEA had no effect on MHC-I expression in COLO 205 cells as determined by flow cytometry, in contrast to the activity of the phytocannabinoids CBD and CBG after 48 hr of treatment. (B) An MHC-I induction time course shows that elevated MHC-I is first detectable at 24 hr in cultured COLO 205 cells treated with 15 µM CBD but has reached maximum expression by 24 hr in cells treated with 3 ng/ml IFN-γ. (C) . Parallel dose titrations spotlight the dramatic impact of IFN-γ and CBD on MHC-I expression in COLO 205 cells. The titrations also clarify differences in potency and dose effect between the cytokine and the cannabinoid. The dose titrations were conducted in triplicate in the same experiment and are representative of numerous IFN-γ and CBD comparisons. Statistical analysis using a two-sided T-test with unequal variances revealed that every titration data point has a p-value of less than 0.005 vs. the untreated sample. MFI: mean fluorescent intensity.

Article Snippet: Relative expression levels of 111 soluble mouse proteins including cytokines, chemokines and growth factors were evaluated using the Proteome Profiler Mouse XL cytokine array kit (R&D System, ARY028) following the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Activity Assay, Cell Culture, Titration

Journal: Frontiers in Immunology

Article Title: Specific cannabinoids revive adaptive immunity by reversing immune evasion mechanisms in metastatic tumours

doi: 10.3389/fimmu.2022.982082

Figure Lengend Snippet: Fold change of cytokine production in Metastatic Carcinomas treated with cannabigerol or in supernatant upon treatment of 0.00875 mg/mL cannabigerol (0.05529 μmol) and 0.1ug/mL (100ng/mL) (5.832x10 -6 nmol) IFN-γ relative to DMSO (vehicle)-treated metastatic A9 cells. (A–C) Fold change of cytokines present on microarray (D) Cytokines implicated in the IL4, 10, and 13 pathways. Data was determined using Proteome Profiler Mouse XL cytokine array kit and Image J protein analyzer add-on. Pathway analysis was determined using Reactome Database release 65, Pathway Brower Version 3.5.9.

Article Snippet: Relative expression levels of 111 soluble mouse proteins including cytokines, chemokines and growth factors were evaluated using the Proteome Profiler Mouse XL cytokine array kit (R&D System, ARY028) following the manufacturer’s instructions.

Techniques: Microarray