N/TERT-TR keratinocytes (KCs) were engineered to express a tetracycline (Tet)-inducible shRNA as described 57 (link). These cells (N/TERT-TR-shTRAF3IP2) were seeded at 8,000 cells cm−2 in the presence or absence of 1 μgxml−1 Tet. When confluent (after 7 days), cells were stimulated for 1 to 4 hours with 200ngxml−1 recombinant human (rh) IL-17A (R&D Systems). RNA was isolated using RNAeasy columns (Qiagen) and reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative real-time polymerase chain reaction (qPCR) was performed using Taqman primers (Life Technologies) specific for nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta (NFKBIZ, Hs00230071-m1), TNF receptor associated factor 3 interacting protein 2 (TRAF3IP2, Hs00974570-m1) and the control gene RPLP0 (Hs99999902_m1). From the same experiments, NFKBIZ and TRAF3IP2/Act1 proteins were assessed by western blotting, utilizing rabbit polyclonal IgG (respectively from Cell Signal Transduction #9244 and Santa Cruz Biotechnology sc-11444, both diluted 1:1000) to detect protein, with β-actin (Cell Signal Transduction #4967 dilution 1:5000) as a loading control. Statistical analysis was performed using GraphPad software (Prizm).The uncropped scans of the blots are shown in Supplementary Fig. 4 .
Recombinant human il 17a
Manufactured by R&D Systems
Sourced in United States
Recombinant human IL-17A is a cytokine produced by activated T cells. It is a member of the IL-17 family and plays a role in the inflammatory response.
Automatically generated - may contain errors
Lab products found in correlation
Taqman primer, by Thermo Fisher Scientific (2 mentions)
Rnaeasy column, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rhtnf α, by R&D Systems (2 mentions)
Il 17f, by R&D Systems (2 mentions)
Hacat cell, by National Collection of Authenticated Cell Cultures (1 mentions)
Hacat cells, by Thermo Fisher Scientific (1 mentions)
Rhil 22, by R&D Systems (1 mentions)
Rhil 1α, by R&D Systems (1 mentions)
Hacat cell line, by National Centre for Cell Science (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Ebm 2 basal medium, by Lonza (1 mentions)
Rat tail collagen type 1, by Thermo Fisher Scientific (1 mentions)
Rhil 6, by R&D Systems (1 mentions)
Rhil 6r, by R&D Systems (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Poly 1 c, by Novus Biologicals (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
22 protocols using recombinant human il 17a
1
Tet-Inducible shRNA for IL-17A Response
2
HaCaT Cell Cytokine Stimulation and GSDME Knockdown
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Human keratinocyte cell line, HaCaT cells, were obtained from China Center for Type Culture Collection (Wuhan, China). HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (both from Gibco, CA, USA) in 5% CO2 environment at 37 °C. HaCaT cells were treated with 10 ng/mL recombinant human (rh) IL-17A (R&D, Minneapolis, USA), 10 ng/mL rh OSM (R&D), 10 ng/mL rh TNF-α (R&D), 10 ng/mL rh IL22 (R&D), and 10 ng/mL rh IL1-α (R&D) in combination for 6, 12, 24, or 48 h. Transfection was performed according to previously described methods (45). For GSDME knockdown, HaCaT cells were transfected with pGLVH1/GFP + Puro lentivirus vector containing NC shRNA (shNC sequence: 5’-TTCTCCGAACGTGTCACGT-3’) or GSDME shRNA (shGSDME sequence: 5’-GCAGAAGTGTGTGATCTCTGA-3’) (GenePharma, Shanghai, China). Stable knockdown HaCaT cells were obtained and selected by puromycin incubation.
3
Tet-Inducible shRNA for IL-17A Response
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N/TERT-TR keratinocytes (KCs) were engineered to express a tetracycline (Tet)-inducible shRNA as described 57 (link). These cells (N/TERT-TR-shTRAF3IP2) were seeded at 8,000 cells cm−2 in the presence or absence of 1 μgxml−1 Tet. When confluent (after 7 days), cells were stimulated for 1 to 4 hours with 200ngxml−1 recombinant human (rh) IL-17A (R&D Systems). RNA was isolated using RNAeasy columns (Qiagen) and reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative real-time polymerase chain reaction (qPCR) was performed using Taqman primers (Life Technologies) specific for nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta (NFKBIZ, Hs00230071-m1), TNF receptor associated factor 3 interacting protein 2 (TRAF3IP2, Hs00974570-m1) and the control gene RPLP0 (Hs99999902_m1). From the same experiments, NFKBIZ and TRAF3IP2/Act1 proteins were assessed by western blotting, utilizing rabbit polyclonal IgG (respectively from Cell Signal Transduction #9244 and Santa Cruz Biotechnology sc-11444, both diluted 1:1000) to detect protein, with β-actin (Cell Signal Transduction #4967 dilution 1:5000) as a loading control. Statistical analysis was performed using GraphPad software (Prizm).The uncropped scans of the blots are shown in Supplementary Fig. 4 .
4
Culturing HaCaT and NHEK Cells
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HaCaT cell line was procured from National Centre for Cell Sciences (NCCS), Pune, India and maintained in DMEM F-12 media containing 10% (v/v) fetal calf serum, 100 Units/ml penicillin, 100mg/ml streptomycin, 0.25mg/ml amphotericin at 37 °C in a humidified atmosphere at 5% CO2. Primary normal human epidermal keratinocytes (NHEK) were isolated from the foreskin samples by 15–18 h treatment with Dispase II at 4 °C followed by trypsinization at 37 °C for 5 mins by 0.1% trypsin. Keratinocytes were cultured in keratinocyte serum free media supplemented with epidermal growth factor and bovine pituitary extract and once 80% confluent, cells were stimulated with recombinant human (rh) IL-17A (R&D System, Minneapolis, MN) at 100 ng/ml for 6 hours before harvesting for further experiments.
5
Inflammatory Cytokine Effects on hCMEC/D3 Cells
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Human cerebral microvascular endothelial cells (hCMEC/D3) (Weksler et al., 2013; Weksler et al., 2005) at passages 26-35 were cultured on rat-tail collagen type I-coated tissue culture plates (for gene expression studies) or Transwell® inserts (for assessments of cell monolayer integrity) containing EBM-2 basal medium (Lonza, Walkersville, MD) supplemented with the EGM-2 SingleQuot reagents obtained from the manufacturer. Cells were maintained at 37 °C and 5% CO2 until confluence. Immortalized hCMEC/D3 cell line was kindly provided by Ashley Hayes, F. Hoffmann-La Roche Ltd., Basel, Switzerland. rat-tail collagen type I was obtained from Gibco, Madison, WI.
Recombinant human (rh) IL17A, rhTNF-α, rhIL6 and rhIL6R were purchased from R&D Systems. The experiments were run in 2-fold serial dilutions for each cytokine. Final concentrations used in this study: 50 ng/ml IL-17A ± 1 ng/ml TNF-α or 25 ng/ml IL-6 + 50 ng/ml IL-6R. For gene expression analyses, total RNA was extracted using RNeasy Mini Kit (QIAGEN) after 2 days of treatment with the cytokines.
Recombinant human (rh) IL17A, rhTNF-α, rhIL6 and rhIL6R were purchased from R&D Systems. The experiments were run in 2-fold serial dilutions for each cytokine. Final concentrations used in this study: 50 ng/ml IL-17A ± 1 ng/ml TNF-α or 25 ng/ml IL-6 + 50 ng/ml IL-6R. For gene expression analyses, total RNA was extracted using RNeasy Mini Kit (QIAGEN) after 2 days of treatment with the cytokines.
6
Keratinocyte Transfection and IL-17A Treatment
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Keratinocytes were isolated from 8-week-old C57BL6/DBA female mouse dorsal skin and immortal keratinocyte cell lines were generated and cultured as previously described28 (link). Keratinocytes were seeded in a 6-well plate to a confluence of about 80% 24 h prior to transfection. Empty expression vector cassette pCDNA4 was used as a negative control. Keratinocytes were transiently transfected with pEGFP-C1-Gilz1 plasmid (500 ng) using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s protocol. Five hours after transfection, medium was replaced and cells were treated with recombinant human IL-17A (R&D Systems) at a concentration of 100 ng/ml for 24 h.
7
Cytokine Stimulation of NHBE Cells
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PolyI:C (high molecular weight) was purchased from Imgenex (San Diego, CA). Recombinant human IL-17A was from R&D Systems (Minneapolis, MN). Concentrations of PolyI:C and IL-17A used in this study were 50 μg/ml and 10 ng/ml, respectively. In our preliminary data and previous report [10 (link)], we have performed a concentration study of PolyI:C treatment (0.1–200 μg/ml) in NHBE cells. Because 50 μg/ml of PolyI:C had potent stimulation in proimflammatory cytokines mRNA expression (data not shown), we chose 50 μg/ml of PolyI:C treatment in the present study. Concerning the dose of IL-17A, we have previously shown that 10 ng/ml of IL-17A induced MUC5AC mRNA expression in NHBE cells, and slight decrease of stimulation was seen when dose higher than 20 ng/ml were used [18 (link)]. Thus, we selected 10 ng/ml IL-17A in this study.
8
Recombinant IL-17A and TGF-β Protocol
9
IL-17 Impact on MSC Osteogenesis
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In vitro IL-17 simulation of the MSCs was conducted according to two distinct protocols.
The MSCs were cultured for 7 days in EM in the presence or absence of 50 ng/mL recombinant human IL-17A (R&D Systems, Minneapolis, MN, USA).
For the differentiation protocol, MSCs were cultured for 21 days in osteogenic medium (OM) containing DMEM:F12 (Gibco, Invitrogen), 15% FBS, 100 U/mL penicillin–streptomycin, 100 nM dexamethasone (Sigma, St. Louis, MO, USA), 5 mM β-glycerophosphate (Sigma) and 50 μg/mL ascorbic acid (Sigma) in the presence or absence of 50 ng/mL recombinant IL-17A (R&D Systems). The medium was replaced every 3–4 days.
The MSCs were cultured for 7 days in EM in the presence or absence of 50 ng/mL recombinant human IL-17A (R&D Systems, Minneapolis, MN, USA).
For the differentiation protocol, MSCs were cultured for 21 days in osteogenic medium (OM) containing DMEM:F12 (Gibco, Invitrogen), 15% FBS, 100 U/mL penicillin–streptomycin, 100 nM dexamethasone (Sigma, St. Louis, MO, USA), 5 mM β-glycerophosphate (Sigma) and 50 μg/mL ascorbic acid (Sigma) in the presence or absence of 50 ng/mL recombinant IL-17A (R&D Systems). The medium was replaced every 3–4 days.
10
Recombinant IL-17A Modulation of Ion Transport
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Recombinant human IL-17A (R&D Systems) was dissolved in 4 mM HCl and used at 50 ng/ml. DIDS (Invitrogen) and niflumic acid (Sigma) were dissolved in DMSO as 1000× stocks and added to the mucosal, chloride-free solution at 100 µM each. NF-κB inhibitor II (JSH-23) was dissolved as a 1000× stock in DMSO and used at 20 µM.
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