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: Medicina (Kaunas, Lithuania)

Article Title: Radiation Induces Bone Microenvironment Disruption by Activating the STING-TBK1 Pathway.

doi: 10.3390/medicina59071316

Figure Lengend Snippet: Figure 3. IR-induced expression changes in the secretion SASP components in a mouse model of bone degenerative injury. (A) The SASP composition in mice serum post-irradiation was detected by Mouse Cytokine microarray membrane analysis. Quantified expression levels of the secretory cytokines, expressed as corrected the pixel density, n = 2. The criteria for defining up- or downregulation was greater or less than 2 SD by the mean value of corrected pixel density in the control group. (B) Heatmap representation of cytokines based on antibody microarray results. Data are expressed as mean ± SD.

Article Snippet: The following reagents were purchased: lipofectamine RNAiMAX (cat. no. 13778100; Thermo Fisher Scientific, Inc., Waltham, MA, USA); opti-MEM (cat. no. 31985062; Gibco, Eggenstein, Germany); STING small interfering RNA (siRNA), negative control (NC)siRNA (GenePharma Co., Ltd., Shanghai, China); STING small-molecule inhibitor, C176 (cat. no. HY-112906; Med-ChemExpress, Princeton, NJ, USA); acid phosphatase, leukocyte (tartrate-resistant acid phosphatase; TRAP) Kit (cat. no. 387A-1KT; SigmaAldrich, St. Louis, MO, USA); mouse cytokine array (cat. no. ARY028; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Expressing, Irradiation, Microarray, Membrane, Control

Journal: Medicina (Kaunas, Lithuania)

Article Title: Radiation Induces Bone Microenvironment Disruption by Activating the STING-TBK1 Pathway.

doi: 10.3390/medicina59071316

Figure Lengend Snippet: Figure 5. STING-P-TBK1 pathway blocking by siRNA alters inflammatory cytokines secretion and osteoclastogenic activity in an in vitro osteocyte model of irradiation. (A) The knockdown efficiency of STING was verified by western blot analyses. (B) After the STING-siRNA knockdown, the protein expression of P-TBK1 in MLO-Y4 cells was evaluated 3 days post-irradiation by western blot analysis. (C) Effect of STING-targeted siRNA intervention on the expression of bone resorption- related protein RANKL. (D) Effect of STING-targeted siRNA intervention on the mRNA expressions of inflammatory cytokines including IL-1α, IL-6, and NF-κB. (E) STING-targeted siRNA intervention on the paracrine regulation of osteoclastogenesis by IR-induced inflammatory cytokine from OCYs. The RAW264.7 cells were co-cultured with the conditioned medium (CM) of irradiated MLO-Y4 cells following STING-siRNA or NC siRNA intervention. The percentage of TRAP+ OCs was quantified (scale bar, 100 µm; magnification, ×100). Data are expressed as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. control), n = 3. NC siRNA: non-STING targeted siRNA interference; STING siRNA: STING targeted siRNA interference. The CM of MLO-Y4 cells was collected at 3 days after 0 Gy or 4 Gy irradiation with STING siRNA or NC siRNA intervention. CM-0 Gy-NC: non- irradiation with NC siRNA intervention; CM-4 Gy-NC: 4 Gy γ-radiation with NC siRNA intervention; CM-0 Gy-SI: non-irradiation with STING-siRNA intervention; CM-4 Gy-SI: 4 Gy γ-radiation with STING-siRNA intervention.

Article Snippet: The following reagents were purchased: lipofectamine RNAiMAX (cat. no. 13778100; Thermo Fisher Scientific, Inc., Waltham, MA, USA); opti-MEM (cat. no. 31985062; Gibco, Eggenstein, Germany); STING small interfering RNA (siRNA), negative control (NC)siRNA (GenePharma Co., Ltd., Shanghai, China); STING small-molecule inhibitor, C176 (cat. no. HY-112906; Med-ChemExpress, Princeton, NJ, USA); acid phosphatase, leukocyte (tartrate-resistant acid phosphatase; TRAP) Kit (cat. no. 387A-1KT; SigmaAldrich, St. Louis, MO, USA); mouse cytokine array (cat. no. ARY028; R&D Systems, Inc., Minneapolis, MN, USA).

Techniques: Blocking Assay, Activity Assay, In Vitro, Irradiation, Knockdown, Western Blot, Expressing, Cell Culture, Control

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

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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: 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: 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: 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: iScience

Article Title: MYC-mediated silencing of miR-181a-5p promotes pathogenic Th17 responses by modulating AKT3-FOXO3 signaling

doi: 10.1016/j.isci.2022.105176

Figure Lengend Snippet:

Article Snippet: The cytokines in the culture supernatants were detected with Mouse IL-17 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) and Mouse IFN-gamma DuoSet ELISA Kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Virus, Recombinant, Adjuvant, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, cDNA Synthesis, SYBR Green Assay, Acid Assay, Microarray, Plasmid Preparation, Software