Journal: Cell reports

Article Title: Comparing HD knockin pigs and mice reveals the pathological role of IL-17.

doi: 10.1016/j.celrep.2023.113443

Figure Lengend Snippet: Figure 1. The differentially expressed genes in the HD KI pig and mouse striatum (A) Volcanic map for differentially expressed transcripts in the striatum of HD KI and wild-type (WT) pigs (left) or mice (right). Red dots represent the significantly higher levels of genes in HD KI animals than WT, including the upregulated 821 genes in pigs and the 447 genes in mice. Green dots represent lower levels of genes in HD KI animals than WT ones, including the downregulated 279 genes in pigs and the 819 genes in mice. Black dots represent genes with no differential expression levels between HD KI and WT animals. (B) Wayne’s diagram shows that there were 267 genes commonly regulated in two species and that 833 and 999 differential genes were species dependent in pigs and mice. (C) Gene networks of different functions via GO categorization in the striatum between HD KI pig and mouse, normalized to their WT controls. The red and bold type indicates ‘‘immune system process’’—an associated cluster that is relevant to inflammatory functions. (D) The genes related to ‘‘IL-17 signaling pathway’’ functions extracted from ‘‘immune system process’’ are shown in the heatmaps. Note that the red and bold types of traf3ip2, il17ra, il17rc, ikbke, ikbkb, and ikbkg genes were upregulated in HD KI pigs as compared to WT pigs but showed no difference between HD KI and WT mouse models (n = 3, 4- to 6-month-old female HD KI and WT mice, n = 3, 4- to 6-month-old female HD KI and WT pigs).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Vinculin Sigma-Aldrich Cat# MAB3574; RRID:AB_2304338 IL-17RA Abcam Cat# ab263908 TRAF3IP2 Protein-tech Cat# 26692-1-AP; RRID:AB_2880606 IKKE CST Cat# 3416T GFAP Abcam Cat# ab7260; RRID:AB_305808 IBA1 Abcam Cat# ab178847; RRID:AB_2832244 SNAP25 Abcam Cat# ab41455; RRID:AB_945552 PSD95 Abcam Cat# ab238135; RRID:AB_2895158 DARPP32 Sigma-Aldrich Cat# D2693-1VL NeuN Abcam Cat# ab177487; RRID:AB_2532109 S100b Abcam Cat# ab52642; RRID:AB_882426 ALDH1L1 Abcam Cat# ab177462 HTT(1C2) Millipore Cat# MAB1574; RRID:AB_1674243 His Sangon, China Cat# D191001; RRID:AB_2940946 Fixable Viability Stain 510 BD Biosciences Cat# 564406; RRID:AB_2869572 anti-human BV421-CD45 BioLegend Cat# 304032; RRID:AB_2561357 anti-human Percp-cy5.5-CD4 BioLegend Cat# 317428; RRID:AB_1186122 anti-human APC-IL-17A BioLegend Cat# 512334; RRID:AB_2563986 anti-mouse BV421-CD45 BioLegend Cat# 103134; RRID:AB_10899570 anti-mouse Percp-cy5.5-CD4 BD Biosciences Cat# 550954; RRID:AB_393977 anti-mouse APC-IL-17A BioLegend Cat# 506916; RRID:AB_536017 Goat pAb to Ms IgG (HRP) Abcam Cat# ab6789; RRID:AB_955439 Goat pAb to Rb IgG (HRP) Abcam Cat# ab6721; RRID:AB_955447 Alexa FluorTM 488 goat anti-rabbit IgG (H + L) Thermo Fisher Cat# A-11034; RRID:AB_2576217 Alexa FluorTM 555 goat anti-rabbit IgG (H + L) Thermo Fisher Cat# A-21429; RRID:AB_2535850 Alexa FluorTM 555 goat anti-mouse IgG (H + L) Thermo Fisher Cat# A-21424; RRID:AB_141780 Chemicals, peptides, and recombinant proteins Recombinant mouse interieukin-17A Sangon, China Cat# C521335-0010 TRIzol Thermo Fisher Cat# 15596018CN PrimeScriptTM RT reagent Kit TaKaRa Cat# RR047A QuantiNova SYBR Green PCR Kit QIAGEN Cat# 208052 BD Perm/Wash buffer BD Biosciences Cat# 554723 BD Cytofix/Cytoperm BD Biosciences Cat# 554722 EBioscience TM RBC Lysis Buffer Thermo Fisher Cat# 00-4333-57 Cell Activation Cocktail (with Brefeldin A) Biolegend Cat# 423303 PBS Hyclone, China Cat# SH30256 DAPI Sigma-Aldrich Cat# D9542 DMEM Gibco Cat# 11320-033 FBS Hyclone, China Cat# SH30084.03 Critical commercial assays DAB substrate kit Solarbio, China Cat# PC0020 Mouse IL-17 ELISA kit Gelatins, China Cat# JLC3574 Pig IL-17 ELISA kit Gelatins, China Cat# JLC9766 Rapid GolgiStainTM Kit FD NeuroTech Cat# PK401 (Continued on next page) 14 Cell Reports 42, 113443, December 26, 2023

Techniques: Quantitative Proteomics

Journal: Journal of Extracellular Vesicles

Article Title: An extracellular vesicular mutant KRAS‐associated protein complex promotes lung inflammation and tumor growth

doi: 10.1002/jev2.12307

Figure Lengend Snippet: EV‐KRAS complex facilitates inflammation through the IL‐17A/FGF21 axis. (a) HE staining of lung tissue from mice (blocked with IL‐17R/Fc or anti‐FGF21 antibody) intratracheally administered extracellular vesicle (EV) KRAS complex (liposome‐EV‐KRAS). (b) ELISA for TNFα, IL‐17A and IL‐6 from lung tissue from mice (blocked with IL‐17R/Fc or anti‐FGF21 antibody) intratracheal administered liposome‐EV‐KRAS. (c) Adenosine triphosphate (ATP) cell proliferation assay showing proliferation of Jurkat cells in response to lung tumour lysate with and without IL‐17A depletion using IL‐17A immunoprecipitation. (d) Luciferase assay for FGF21 promotor assessment, TC1 cells treated with respective lung tumour lysate with and without IL‐17A depletion using IL‐17A immunoprecipitation. (e) Western blot for PI3K, SIRT1, NFκB‐ TC1 cells treated with respective lung tumour lysate with and without FGF21 depletion using FGF21 immunoprecipitation. (f) qRT‐PCR for genes involved in EMT pathway‐ TC1 cells treated with respective lung tumour lysate with and without FGF21 depletion using FGF21 immunoprecipitation. For in vivo treatment mice were administered with 50 μl of 10 × 1010 liposome particles/ml and for in vitro treatment 2 × 108 liposome particle/ml for respective group were used. Data are mean ± SEM from three replicates using one‐way ANOVA, ** p < 0.01.

Article Snippet: In vivo cytokine neutralization experiments involved administering mAbs against FGF21 (Abclonal, Woburn, MA) and recombinant Mouse IL‐17RA/IL‐17R Fc Chimera Protein (R&D System) intraperitoneally.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Immunoprecipitation, Luciferase, Western Blot, Quantitative RT-PCR, In Vivo, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

doi: 10.3390/ijms19103089

Figure Lengend Snippet: Screening of the interleukin 17 cognate receptor A (IL-17RA)-binding variants in enzyme-linked immunosorbent assay (ELISA). Bacterial cell lysates of individual ARS and ARU clones were screened for binding to the immobilized recombinant IL-17RA receptor. The binding proteins were produced in the form of in vivo biotinylated ARS/ARU-TolA-AVI fusion proteins and their binding to IL-17RA was visualized by streptavidin-HRP conjugate.

Article Snippet: Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 μg/mL, produced in E. coli SHuffle strain) or 5 μg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R&D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Recombinant, Produced, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

doi: 10.3390/ijms19103089

Figure Lengend Snippet: Comparison of a sequence similarity of the selected ARS and ARU clones. Similarity tree of polypeptide sequences of 51 analyzed ARS and ARU variants that were targeted to the recombinant extracellular IL-17RA receptor and selected by ribosome display. Sequence analysis of the ARS/ARU binders revealed 17 unique sequence variants that were clustered into 7 sequence families. For a similarity analysis, only the sequences between residues 20 and 46 were compared, as the N-terminal amino acid positions 1–19 were non-randomized.

Article Snippet: Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 μg/mL, produced in E. coli SHuffle strain) or 5 μg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R&D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight.

Techniques: Comparison, Sequencing, Clone Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

doi: 10.3390/ijms19103089

Figure Lengend Snippet: Binding of the selected representatives of ARS sequence families to the immobilized human IL-17RA-IgG chimera in ELISA. Purified binding proteins were produced in the form of in vivo biotinylated His 6 -ARS-TolA-AVI fusion proteins. Binding to IL-17RA-IgG was visualized by streptavidin-HRP conjugate. Each point represents the mean value ± standard deviation (SD).

Article Snippet: Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 μg/mL, produced in E. coli SHuffle strain) or 5 μg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R&D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight.

Techniques: Binding Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Purification, Produced, In Vivo, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

doi: 10.3390/ijms19103089

Figure Lengend Snippet: ARS ligands compete with IL-17A cytokine for binding to the human IL-17RA-IgG receptor chimera. The IL-17RA-IgG chimera was immobilized on an ELISA plate and serially diluted inhibitory His 6 -ARS-TolA-AVI ligands were used to compete for binding with 10 nM IL-17A. Bound IL-17A was detected with anti-IL-17A polyclonal antibody in combination with secondary anti-IgG-HRP conjugate. His 6 -ABDwt-TolA-AVI served as a negative control. Error bars represent standard deviations (SDs).

Article Snippet: Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 μg/mL, produced in E. coli SHuffle strain) or 5 μg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R&D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

doi: 10.3390/ijms19103089

Figure Lengend Snippet: Binding of the ARS variants to human THP-1 cells. ( A ) The expression of IL-17RA on the surface of THP-1 cells was confirmed by anti-IL-17RA antibody. ( B ) For binding assay, 2.5 × 10 5 THP-1 cells were incubated with in vivo biotinylated His 6 -ARS-TolA-AVI proteins or His 6 -ABDwt-TolA-AVI negative control (10 µg/mL) for 30 min at 4 °C. The cell-bound proteins were stained with streptavidin-PE for 30 min at 4 °C and analyzed by flow cytometry. Each bar represents the mean value ± SD of three independent experiments.

Article Snippet: Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 μg/mL, produced in E. coli SHuffle strain) or 5 μg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R&D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight.

Techniques: Binding Assay, Expressing, Incubation, In Vivo, Negative Control, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

doi: 10.3390/ijms19103089

Figure Lengend Snippet: Modeling of ARS/IL-17RA interactions by docking. Summary of the first three poses of the ARS004 ( A ), ARS012 ( B ), ARS019 ( C ), and four poses of the ARS043 ( D ) binding to the complex of IL-17RA (brown) and dimer of human IL-17A (green/cyan), in decreasing predicted order of probability demonstrated in red (the most probable), orange, yellow and magenta, respectively.

Article Snippet: Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 μg/mL, produced in E. coli SHuffle strain) or 5 μg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R&D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

doi: 10.3390/ijms19103089

Figure Lengend Snippet: Structural effect of amino acid alterations in the ABD scaffold. ( A ) The most probable pose of the ARS004/IL-17RA interaction obtained by flexible side chain docking (red, see also A) compared to the structure relaxed by 100 ns molecular dynamics simulation ( blue ). ( B ) Modeling of the ARS014/IL-17RA interaction by docking. The initial geometry of ARS014 was obtained from a 1 µs molecular dynamics simulation. Summary of the first three poses of ARS014 binding to the IL-17RA ( brown ) is shown in decreasing predicted order of probability demonstrated in red (the most probable), orange, and yellow, respectively. ( C ) The third most probable pose ( yellow ) of the ARS014/IL-17RA interaction compared to the structure relaxed by 100 ns molecular dynamics simulation ( blue ).

Article Snippet: Polysorp plate (NUNC, Roskilde, Denmark) was coated by IL-17RA recombinant protein (10 μg/mL, produced in E. coli SHuffle strain) or 5 μg/mL human IL-17RA-IgG chimera (Recombinant Human IL-17 RA/IL-17 R Fc Chimera Protein, R&D Systems, Minneapolis, MN, USA) in coating buffer (100 mM bicarbonate/carbonate solution, pH = 9.6) at 7 °C overnight.

Techniques: Binding Assay