Recombinant human il 33

MSCV-LTRmiR30-PIG (LMP) is a retroviral vector designed for the dual expression of GFP and short hairpin RNAs (shRNA) (Open Biosystems). SETD2 shRNA#1 and SETD2 shRNA#2 were generated by ligation of synthesized DNA oligonucleotides targeting coding regions of human SETD2 to the LMP vector. Phoenix cells were transfected with retroviral plasmids and the packaging plasmid 10A1 using polyethylenimine (PEI, Polysciences). Viral supernatant was collected after transfection. FACS-sorted human peripheral blood Treg cells (CD4⁺CD127⁻CD25⁺ cells) were cultured in α-CD3 (2 ug/ml, eBioscience)/α-CD28 (2 ug/ml, eBioscience) coated 96-well plate with RPMI 1640 complete medium containing recombinant human IL-2 (10 ng/ml, Peprotech), recombinant human IL-33 (10 ng/ml, Peprotech), α-human-IFN-γ (4 ug/ml, BD biosciences) for 5–8 days. Then, spin-infection was performed at 2500 rpm for 1.5 h at 30 °C and cultured with an additional 24 h in an incubator with 5% CO₂, in virus supernatant containing 8 ug/ml polybrene (Sigma-Aldrich), α-CD3, α-CD28, IL-2, IL-33, and α-IFN-γ. Cells were analyzed 36–48 h later after the second round of spin infection. The target sequences of SETD2 are: #1 5′-ACTCACGGTGTTATGAATAAG-3′; #2 5′-TGTCTGGAACTCATACAGAAC-3′.

Reagents used in this study were acquired from the indicated suppliers: trans-resveratrol, cyclodextrin, 5Z-7-Oxozeaenol and Evans blue (Sigma-Aldrich, St. Louis, MO, USA); LY294002, wortmannin, and ICI 182,780 (Abcam, Cambridge, UK); PF-3644022 (TOCRIS Bioscience, Bristol, U.K.); recombinant mouse IL-3, recombinant mouse stem cell factor (SCF), and recombinant human IL-33 (PeproTech, Rocky Hill, NJ, USA); recombinant human IL-3 (Thermo Fisher Scientific, Wilmington, DE, USA); recombinant mouse IL-33 (R & D systems, Minneapolis, MN, USA); anti-TNP IgE, anti–DNP mouse IgE mAb, anti–mouse CD16/32, PE–conjugated anti–mouse c-kit Ab, and APC–conjugated anti–mouse ST2 Ab (BD Bioscience, San Jose, CA, USA); DNP–BSA (Cosmo Bio, Tokyo, Japan); APC–conjugated anti–mouse CD63 Ab (Miltenyi Biotec, Bergisch Gladbach, Germany); anti–phospho–transforming growth factor β-activated kinase 1 (TAK1) Ab (Thr184/187; #4508), anti–phospho–IκB kinase (IKK) α/β Ab (Ser176/177; #2078), anti–phospho–p65 Ab (Ser536; #3033), anti–phospho–p38 Ab (Thr180/Thy182; #4511), anti–phospho–MK2 Ab (Thr334; #3007), anti–phospho–Akt Ab (Ser473; #4060), anti–phospho–Gab2 Ab (Tyr452; #3882), anti–phospho–Syk Ab (Tyr525/526; #2710), anti–phospho–p70S6K Ab (Tyr389; #9234), anti–phospho–AMPK Ab (Thr172; #2535), anti–β-actin Ab (#4970), and AICAR (#9944) (Cell Signaling Technology, Danvers, MA, USA).

Human THP-1 cell line was from Sigma-Aldrich and recombinant human IL-33 was supplied by Peprotech. Antibodies were from Cell Signaling Technology [anti-p44/p42 (ERK1/2) (9102), anti-p38 (9212), anti-JNK1/2 (9252), NF-κB p105/p50 (3035)], Santa Cruz Biotechnology [NF-κB p65 (sc-372), β-Actin (sc-130656)] or Sigma-Aldrich [β-Actin (A2228)].

Recombinant human IL-33 was purchased from Pepro Tech (Rocky Hill, NJ, USA). The mouse monoclonal anti-human MCP-1 antibody was from Santa Cruz Biotechnology (Heidelberg, Germany). The rabbit polyclonal antibodies for JNK, phospho-JNK (Thr183/Tyr185), p38, phospho-p38 (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (p42/44 MAPK), c-Jun, and phospho-c-Jun (Ser73) were obtained from Cell Signaling Technology (Beverly, MA, USA). SP600125 (JNK inhibitor) was purchased from BIOMOL (Plymouth Meeting, PA, USA). Simvastatin, PD98059 (ERK1/2 inhibitor), and SB203580 (p38 MAPK inhibitor) were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Mevalonate was obtained from Sigma (St Louis, MO, USA).

Human mast cells HMCs-1 were obtained from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Science (TCHu69, Shanghai, China) and cultured in DMEM medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 mg/ml streptomycin sulfates at 37°C with 5% CO₂. HMCs-1 were seeded into 24-well culture plates at 2 × 10⁵ cells/well; 0.4 μg/mL recombinant human IL-33 (Peprotech, Princeton, USA) was added at the same time, while an equal amount of DMEM medium was added as blank control. After 24 h of stimulation, cells were harvested and processed for further experiments. Experiments were assayed in technical and biological triplicate.