Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: (A) RT-PCR amplification of IL-9 and IL-33 from rhesus PBMC and CD4+ T cells. (B) Expression of IL-9 by CD4+ T cells. PBMC from two rhesus macaques were stimulated with PMA and ionomycin for four hours, permeabilized, stained with anti-human IL-9 antibody, and analyzed by flow cytometry. Plots are gated on live, CD19-CD20-CD8-CD3+CD4+ T cells. Nucleotide alignment of rhesus observed (Rmobsv), predicted (Rmpre), mouse, and human (Hum) IL-9 (C) and IL-33 (D). The boxes indicate codon differences between the rhesus observed and human.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Staining, Flow Cytometry

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: Amino acid alignments of mouse, human, predicted, and synthesized (syn) IL-9 (A) and IL-33 (B) proteins.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Synthesized

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: HEK293T cells were transfected with cloned rhesus IL-9 and IL-33 and supernatant was collected to measure the production of IL-9 (A) and IL-33 (B) protein by ELISA. Decreasing titrations of recombinant human cytokine included as positive control, supernatant from empty vector control transfected cells (pUNO1) included as negative control. Bars represent mean ± SEM of triplicate samples.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Transfection, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Plasmid Preparation, Negative Control

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: (A) Human MO7e cells were cultured with rhesus macaque IL-9 (RmIL-9), in the absence or presence of IL-9 neutralizing antibody for 3 days. (B) Mouse D10.G4.T1 cells were cultured with rhesus macaque IL-33 (RmIL-33) in the absence or presence of IL-33 neutralizing antibody for 3 days. Recombinant human IL-9 and IL-33 were used as positive control and empty vector pUNO1 as negative control. Bars represent mean ± SEM of triplicate samples. * indicates p<0.05 compared to pUNO1.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Cell Culture, Recombinant, Positive Control, Plasmid Preparation, Negative Control

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: Rhesus BLCL-C162 cells were cultured for 3 days with rhesus macaque IL-9 or IL-33 in the presence or absence of neutralizing antibody. * indicates p<0.05 compared to pUNO1.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Cell Culture

Journal: Cell Reports

Article Title: Mechanical force receptor Piezo1 regulates TH9 cell differentiation

doi: 10.1016/j.celrep.2024.115136

Figure Lengend Snippet: Figure 6. Piezo1 deficiency inhibits TH9 cell differentiation through the SIRT3-SDHA-OXPHOS metabolism pathway (A) The oxygen consumption rate (OCR) was examined as a readout for OXPHOS in CD4+ T cells isolated from WT and Piezo1/ mice for 5 days under TH9 cell- inducing conditions. (B) Mean fluorescent intensity (MFI) of SDHA in CD4+ T cells isolated from WT and Piezo1/ mice for 5 days under TH9 cell-inducing conditions. (C) SDH/complex II activities of T cells isolated from WT and Piezo1/ mice for 5 days under TH9 cell-inducing conditions by ELISA. (D) Mass spectrometry analysis of the metabolite abundance of fumarate (FA), succinate (SC), and malate (MA) in CD4+ T cells isolated from WT and Piezo1/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GsMTx4 MedChemExpress, USA Cat#HY-P1410 Ionomycin Sigma-Aldrich, USA Cat#I0634 Yoda1 MedChemExpress, USA Cat#HY-18723 Critical commercial assays Co-IP buffer Thermo Fisher Scientific, USA Cat#88804 CD4+ T cell isolation kit Miltenyi Biotec, USA Cat#130-104-453 ECL kit Beyotime Biotechnology, China Cat#P10018AS Enhanced BCA protein assay kit Beyotime Biotechnology, China Cat#P0009 Foxp3/transcription factor staining buffer set BD Bioscience, USA Cat#560133 Mito stress test kit Seahorse Bioscience, USA Cat#101706-100 NE-PER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific, USA Cat#78835 RIPA lysis buffer Beyotime Biotechnology, China Cat#P0013B RNeasy mini kit Qiagen, Germany Cat#74106 PrimeScriptTM RT reagent kit TakaRa, Japan Cat#TakaRa_RR037A SuperReal preMix plus SYBR Green Tiangen, Germany Cat#FP205-02 Dual-Luciferase assay system Promega, USA Cat#E1910 Mouse IL-9 DuoSet ELISA R&D system, USA Cat#DY409 Human IL-9 DuoSet ELISA R&D system, USA Cat#DY209-05 Deposited data Raw and analyzed data This paper GEO:GSE268658 Experimental models: Cell lines Mouse melanoma cell line B16.F10 China cell bank ATCC, China B16F10 Mouse lymphoma cell line EL-4 China cell bank ATCC, China EL4 HEK293T ATCC, USA Cat#CRL-11268 Phoenic-Eco packaging cells Allele Biotechnology, USA Cat#ABP-RVC-10001 Experimental models: Organisms/strains Mouse: C57BL/6J (CD45.1) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J (CD45.2) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J-Rag1 / GemPharmatech, China Cat#T004753 Mouse: Piezo1flox/flox GemPharmatech, China Cat#T009627 Mouse: Cd4-Cre GemPharmatech, China Cat#T067676 Mouse: Piezo1 / GemPharmatech, China Cat#T012739 Mouse: Sirt3flox/flox GemPharmatech, China Cat#T006658 Mouse: Hif1aflox/flox GemPharmatech, China Cat#T007329 Mouse: C57BL/6J (GFP) GemPharmatech, China Cat#T054573 Oligonucleotides siRNA targeting sequence: SDHA siRNA, 50-UAAAUCUUCCCAUCUUCAGUU-30 This paper NA siRNA targeting sequence: Piezo1 siRNA, 50-CACCGGCATCTACGTCAAATA-30 This paper NA Piezo1 (Mm01241549_m1) Applied Biosystems, USA Cat#4331182 Piezo1 (Hs00207230_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Mm00434305_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Hs00174125_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Mm00452131_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Hs00202030_m1) Applied Biosystems, USA Cat#4331182 (Continued on next page) Cell Reports 44, 115136, January 28, 2025 17

Techniques: Cell Differentiation, Isolation, Enzyme-linked Immunosorbent Assay, Mass Spectrometry

Journal:

Article Title: Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris -infected mice

doi:

Figure Lengend Snippet: Induction of IL-9 specific autoantibodies. (A) Vaccination against IL-9. Groups of four C57BL/6 mice were injected subcutaneously with IL-9-OVA complexes in CFA (IL-9-OVA), with noncomplexed IL-9 and OVA (IL-9 + OVA) or with CFA alone (CFA). Boosts were carried out in IFA after 2 and 4 wk. Serially diluted sera, collected 2 wk after the last immunization, were tested for IL-9 inhibition in a TS1 cell proliferation assay. Cell growth was evaluated by measuring hexosaminidase activity. Means ± SD are indicated. (B) Specificity of anti-IL-9 activity. Pools of 1/320 diluted sera from immunized (IL-9-OVA) or control (CFA) mice were tested for growth inhibition of TS1 cells, in the presence of either natural mIL-9, baculovirus-derived mIL-9 (rec.), or mIL-4. Cell proliferation was measured after 3 days of culture. Results are given as means ± SD.

Article Snippet: IL-9-OVA complexes were detected in column fractions by ELISA by using 2C12, a hamster monoclonal anti-IL-9 antibody produced in our laboratory for capture, and a rabbit anti-OVA antiserum followed by peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) for detection of complexes.

Techniques: Injection, Inhibition, Proliferation Assay, Activity Assay, Derivative Assay

Journal:

Article Title: Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris -infected mice

doi:

Figure Lengend Snippet: Production of anti-IL-9 autoantibodies in different mouse strains. Eight-week-old C57BL/6, FVB, BALB/c, NOD, DBA/2, or NZW mice were injected with IL-9-OVA complexes (four mice) or adjuvant only (four mice), as described in Materials and Methods. Serially diluted sera, collected 2 wk after the last immunization, were tested in a TS1 cell proliferation assay. Inhibition titers, given for each mouse serum, correspond to serum dilutions inhibiting IL-9-induced cell proliferation by 50%.

Article Snippet: IL-9-OVA complexes were detected in column fractions by ELISA by using 2C12, a hamster monoclonal anti-IL-9 antibody produced in our laboratory for capture, and a rabbit anti-OVA antiserum followed by peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) for detection of complexes.

Techniques: Injection, Proliferation Assay, Inhibition

Journal:

Article Title: Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris -infected mice

doi:

Figure Lengend Snippet: Persistence of the anti-IL-9 response in immunized mice. Three 8-wk-old C57BL/6 mice were injected s.c. with IL-9-OVA complexes in CFA. Mice were boosted with IFA after 2 and 4 wk. Serum was collected 2, 4, 6, 8, 12, 26, and 56 wk after priming and tested for anti-IL-9 activity in a TS1 cell proliferation assay. Results are given as the inhibition titer for individual mouse sera.

Article Snippet: IL-9-OVA complexes were detected in column fractions by ELISA by using 2C12, a hamster monoclonal anti-IL-9 antibody produced in our laboratory for capture, and a rabbit anti-OVA antiserum followed by peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) for detection of complexes.

Techniques: Injection, Activity Assay, Proliferation Assay, Inhibition

Journal:

Article Title: Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris -infected mice

doi:

Figure Lengend Snippet: Inhibition of IL-9-induced mast-cell activation and eosinophilia in immunized mice. Seven-week-old C57BL/6 mice subjected to the IL-9-OVA immunization protocol or immunized with noncoupled IL-9 and OVA or left untreated (five mice per group) were injected i.p. with 107 IL-9-producing TS1.G6 cells, 5 mo after the last boost. Five age-matched mice served as negative controls. All mice were tested 25 days later for MMCP-1 serum levels by ELISA (A), and blood leukocytes were isolated for eosinophil counting (B). Eosinophil percentages were determined by enumerating 20,000 cells per slide. Means ± SEM are indicated.

Article Snippet: IL-9-OVA complexes were detected in column fractions by ELISA by using 2C12, a hamster monoclonal anti-IL-9 antibody produced in our laboratory for capture, and a rabbit anti-OVA antiserum followed by peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) for detection of complexes.

Techniques: Inhibition, Activation Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation

Journal:

Article Title: Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris -infected mice

doi:

Figure Lengend Snippet: Altered response to T. muris in IL-9-OVA-vaccinated mice. (A) Failure of IL-9-OVA-immunized C57BL/6 mice to expel T. muris. C57BL/6 mice immunized with IL-9-OVA complexes or vehicle (eight mice per group) were infected by oral gavage of 200 T. muris eggs. Worms were counted in ceca on day 34 after infection, and results are given as worm burden in each individual. P value between groups is 0.001. On day 13, the worm burden, assessed to verify infectivity, was similar in all animals. (B) Inhibition of T. muris-induced blood eosinophilia in IL-9-vaccinated mice. Blood eosinophils were counted 23 days after infection from infected control or IL-9-vaccinated C57BL/6 mice or uninfected naive animals. Eosinophil percentages were determined by enumerating 500 cells per slide. Results are presented as the mean percentage of blood eosinophils ± SEM (four mice per group). P value is 0.029 for the difference between OVA and IL-9-OVA groups.

Article Snippet: IL-9-OVA complexes were detected in column fractions by ELISA by using 2C12, a hamster monoclonal anti-IL-9 antibody produced in our laboratory for capture, and a rabbit anti-OVA antiserum followed by peroxidase-conjugated anti-rabbit antibody (Santa Cruz Biotechnology) for detection of complexes.

Techniques: Infection, Inhibition