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Human ifnα

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Human IFNα is a recombinant protein that functions as an interferon-alpha, a type of cytokine involved in the immune response. It is commonly used in research and laboratory settings.

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Lab products found in correlation

Mycoalert mycoplasma detection kit, by Lonza (2 mentions) Anti tubulin antibody, by Santa Cruz Biotechnology (2 mentions) Anti gapdh antibody, by Proteintech (2 mentions) Mouse il 1β, by Thermo Fisher Scientific (2 mentions) Poly 1 c hmw, by InvivoGen (2 mentions) Rb anti myc, by Cell Signaling Technology (2 mentions) Ms anti myc, by Cell Signaling Technology (2 mentions) Rb anti actin, by Merck Group (2 mentions) Ms anti flag, by Merck Group (2 mentions) Rb anti 14 3 3, by Santa Cruz Biotechnology (2 mentions) Ms anti spir 1, by Santa Cruz Biotechnology (2 mentions) Ms anti spir 1, by Abcam (2 mentions) Rb anti ddx3, by Cell Signaling Technology (2 mentions) Rb anti ha, by Merck Group (2 mentions) Ms anti gapdh, by Merck Group (2 mentions) Rb anti irf3, by Cell Signaling Technology (2 mentions) Rb anti phospho irf3 ser396, by Cell Signaling Technology (2 mentions) Irdye 680rd conjugated goat anti rabbit igg or anti mouse igg, by LI COR (2 mentions) Irdye 800cw conjugated goat anti rabbit igg or anti mouse igg, by LI COR (2 mentions) Anti c myc agarose, by Santa Cruz Biotechnology (2 mentions)

13 protocols using human ifnα

1

Culturing Human Beta Cell Line and Islets

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The human beta cell line EndoC-βH113 (kindly provided by Dr. R. Scharfmann, University of Paris, France) was cultured in Matrigel fibronectin-coated plates as previously described.14 (link) The MycoAlert Mycoplasma Detection kit (Lonza, Basel, Switzerland) was used to regularly assure that the cells were free from mycoplasma infection.
Human islets from 15 non-diabetic organ donors (see Table S1; beta-cell purity was determined by immunohistochemistry for insulin, as described5 (link)) were isolated in Pisa, Italy, based on methods previously developed15 (link) with the approval of the local ethics committee and sent to Brussels for dispersion and experiments.7 (link)All experiments with EndoC-βH1 cells or human islets are shown as independent biological data (i.e. considering EndoC-βH1 cells from different passages or human islets from different donors as n = 1). Cells were treated with TYKiA and TYKiB provided by Nimbus Lakshmi (Cambridge, MA, USA), the JAK1/2 inhibitor Baricitinib (Selleckchem, Munich, Germany), used as a positive control, and human IFNα (PeproTech, Rocky Hill, NJ, USA) 2000 U/mL (concentration selected based on our previous studies6 (link)).
de Brachène A.C., Castela A., de Beeck A.O., Mirmira R.G., Marselli L., Marchetti P., Masse C., Miao W., Leit S., Evans-Molina C, & Eizirik D.L. (2020). Preclinical evaluation of tyrosine kinase 2 inhibitors for human beta-cell protection in type 1 diabetes. Diabetes, obesity & metabolism, 22(10), 1827-1836.
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2

Protein and Inhibitor Use in Cell Signaling

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The following recombinant proteins and chemical inhibitors were used: Human FGF7 (100‐19, PeproTech Inc., Rocky Hill, NJ or R&D Systems, Minneapolis, MN), human FGF10 (100‐26, PeproTech Inc.), human FGF2 (100‐18, PeproTech Inc.), human IFN‐α (300‐02AA, PeproTech Inc), FGFR1/2/3 inhibitors AZD4547 (S2801, Selleckchem, Houston, TX) and BGJ398 (NVP‐BGJ398; S2183, Selleckchem), PI3K inhibitor LY294002 (InvivoGen, San Diego, CA), AKT1/2 inhibitor A6730 (A6730, Sigma, Munich, Germany), Mek1/2 inhibitor U0126 (662005, Calbiochem, San Diego, CA), PLC‐γ inhibitor U73122 (1278, Tocris, Bristol, UK), and proteasome inhibitors MG132 (S2619 Selleckchem), bortezomib (PS‐341) (S1013, Selleckchem), and epoxomicin (E3652, Sigma).
Maddaluno L., Urwyler C., Rauschendorfer T., Meyer M., Stefanova D., Spörri R., Wietecha M., Ferrarese L., Stoycheva D., Bender D., Li N., Strittmatter G., Nasirujjaman K., Beer H., Staeheli P., Hildt E., Oxenius A, & Werner S. (2020). Antagonism of interferon signaling by fibroblast growth factors promotes viral replication. EMBO Molecular Medicine, 12(9), e11793.
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3

Cell Viability Assay Protocol

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HN4 and HN30 cells were seeded in 96-well plates at a density of 3 × 103 cells per well. Human IFNα (PeproTech, Rocky Hill, NJ, USA), erlotinib (Selleck, Houston, TX, USA), nimotuzumab (Biotech Pharma, Beijing, China) and fludarabine (Selleck) were administrated at the indicated concentrations after cells had adhered. After a 72 h incubation, 20 μl of MTT were added to every well and incubated for 4 h. Then, 200 μl of DMSO were added to dissolve the formazan crystals in each well. The optical density (OD) was measured at 490 nm within 10 min.
Ma H., Jin S., Yang W., Zhou G., Zhao M., Fang S., Zhang Z, & Hu J. (2018). Interferon-alpha enhances the antitumour activity of EGFR-targeted therapies by upregulating RIG-I in head and neck squamous cell carcinoma. British Journal of Cancer, 118(4), 509-521.
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4

Expression and Truncation of Antiviral Protein mViperin

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Total RNA was extracted from Marc-145 cells treated with 3000 U/mL human IFN-α (Peprotech) using QIAprep viral RNA minikit (Qiagen, Hilden, Germany) and cDNA synthesis was performed with SuperScript III Reverse Transcriptase (Invitrogen, Shanghai, China). The full-length mViperin gene was amplified with a set of primers (Table 1), and amplicons were cloned into a pEASY-Simple Blunt vector (Beijing TransGen Biotech Co. Ltd., Beijing, China) and sequenced. The determined nucleotide sequence of mViperin was compared to that found in the database (GenBank accession number: JQ437826.1). To generate the expression vector, the mViperin gene was amplified from a previously sequenced plasmid using the primers shown in Table 1. Polymerase chain reaction (PCR) products were digested with restriction enzymes and cloned into a pVAX-1 vector with the kozak sequence at the N terminus and a FLAG tag at the C terminus to produce a pVAX-mVIP plasmid. Truncations of mViperin were subcloned from the pVAX-mVIP plasmid with a FLAG tag at the N-terminus to produce pVAX-mVIP (5’Δ8, 5’Δ10, 5’Δ12, 5’Δ17, 5’Δ33 and 3’Δ143) plasmids.
Fang J., Wang H., Bai J., Zhang Q., Li Y., Liu F, & Jiang P. (2016). Monkey Viperin Restricts Porcine Reproductive and Respiratory Syndrome Virus Replication. PLoS ONE, 11(5), e0156513.
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5

Regulation of HBV Infection by TRIM14

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Cells of the HepG2 and HEK293T cell lines were maintained in Dulbecco’s Modified Eagle’s Medium containing 10% inactivated fetal bovine serum. All cell lines were maintained in penicillin (100 IU/mL) and streptomycin (100 mg/mL) in 5% CO2 at 37°C. The expression constructs of TRIM14 and STAT1 were generated by cloning the sequence of the coding region into a VR1012 expression vector. Site-directed mutagenesis of STAT1 was generated by QuikChange PCR (TransGen, Beijing, China). The pHBV1.3 plasmids were provided by Dr. Lishan Su, University of North Carolina. Human interleukin (IL)-27 recombinant protein was obtained from R&D Systems (Minneapolis, MN, USA). Human IFN-α was purchased from Peprotech (Jiangsu, China). STAT1, p-STAT1(Tyr701), STAT3, and p-STAT3(Tyr705) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). HBsAg antibody was obtained from Thermo (Shanghai, China), HBc antibody was purchased from Abcam (Shanghai, China), anti-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH antibody was obtained from Proteintech. Antibodies against TRIM14 and IFNAR1 were purchased from Abcam (Shanghai, China).
Tan G., Xu F., Song H., Yuan Y., Xiao Q., Ma F., Qin F.X, & Cheng G. (2018). Identification of TRIM14 as a Type I IFN-Stimulated Gene Controlling Hepatitis B Virus Replication by Targeting HBx. Frontiers in Immunology, 9, 1872.
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6

Quantification of Human IFN-α and IL-6 by ELISA

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human IFN-α and interleukin 6 (IL-6) were quantified by ELISA 24 hours after infection. The following antibodies were used: capture: rat anti-human IL-6 (Pharmingen, 554543) or anti-human IFN-α coating antibody (Bender, 2010-10), detection: biotin rat anti-human IL-6 (Pharmingen, 554546) followed by streptavidin-POD conjugate (Roche, 11089153001) or anti-human-IFN-α HRP-conjugate (eBioscience, BM216MSTK). As standards, recombinant human IL-6 (Peprotech, 200-06) or human IFN-α (Peprotech, 300-02A) were used. HRP-substrate solution contained 1 mg/ml OPD (Sigma, P7288) and 0.03% hydrogen peroxide in substrate buffer (38 mM citric acid, 66 mM disodium hydrogen phosphate). Synthetic DNA oligonucleotide with a CpG motive (CpG 2216) (TIB Molbiol) was used as control for TLR activation. Absorbance values were detected with EMax-plate photometer (Molecular Devices) with SoftMaxPro V5-Sofware.
Dorna J., Kaufmann A., Bockmann V., Raifer H., West J., Matrosovich M, & Bauer S. (2022). Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs. Frontiers in Immunology, 13, 827760.
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7

Culturing Human Pancreatic Beta Cells and Islets

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The human beta cell line EndoC-βH1 (kindly provided by Dr. R. Scharfmann, University of Paris, France) was cultured in Matrigel-fibronectin-coated plates as described [4 (link)]. These cells are free from mycoplasma infection, as evaluated by MycoAlert Mycoplasma Detection kit (Lonza, Basel, Switzerland).
Isolation of human islets from 3 non-diabetic organ donors (ESM Table 1) was performed in accordance with the local Ethical Committee in Pisa, Italy. After arrival in Brussels, islets were dispersed and cultured as in [4 (link)]. All experiments shown with EndoC-βH1 cells or human islet cells (indicated as “n” in the figures) refer to independent biological data (i.e using EndoC-βH1 cells from different passages or human islets from different donors). Where indicated, cells were treated with human IFNα (PeproTech Inc., Rocky Hill, NJ) 20 or 1000 U/ml [4 (link)]. Cells were treated with ruxolitinib (kindly provided by Calibr, CA, USA), cerdulatinib (Selleckchem, Germany), Bayer-18 (Synkinase, UK), or cycloheximide (Sigma-Aldrich, Germany) as indicated.
de Brachène A.C., Dos Santos R.S., Marroqui L., Colli M.L., Marselli L., Mirmira R.G., Marchetti P, & Eizirik D.L. (2018). IFNα induces a preferential long-lasting expression of MHC class I in human pancreatic beta cells. Diabetologia, 61(3), 636-640.
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8

Immunoblotting Protocols Using Diverse Antibodies

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Primary antibodies used were from the following sources: rabbit (Rb) anti-Myc (Cell Signaling, 2278), mouse (Ms) anti-Myc (Cell Signaling, 9B11), Rb anti-actin (Sigma, A2066), Ms anti-FLAG (Sigma F1804), Rb anti-14-3-3 (Santa Cruz, sc-629), Ms anti -Spir-1 (Santa Cruz, sc-517039), Ms anti-Spir-1 (Abcam, ab57463), Rb anti-DDX3 (Cell Signaling, 2635), Rb anti-IKKβ (Cell Signaling, 2684), Rb anti-HA (Sigma, H6908), Ms anti-α-tubulin (Millipore, 05–829), Ms anti-GAPDH (Sigma, G8795), Rb anti-IRF3 (Cell Signaling, 4962), Rb anti-IRF3 (Santa Cruz, SC-9082), Ms anti-COPε (Santa Cruz, sc-133194), Rb anti-phospho-IRF3 Ser396 (Cell Signaling, 4947S) and Rb polyclonal anti-C6 [53 (link)]. For dilutions used for the primary antibodies, see S3 Table. Secondary antibodies used (1:10,000 dilution) were IRDye 680RD-conjugated goat anti-rabbit IgG or anti-mouse IgG and IRDye 800CW-conjugated goat anti-rabbit IgG or anti-mouse IgG (LI-COR).
Reagents used in this study were: anti-c-Myc agarose from Santa Cruz Biotechnology, and monoclonal anti-HA-agarose, clone HA-7, ANTI-FLAG M2 affinity gel and Poly-D-lysine hydrobromide (all from Sigma Aldrich). Human IFNα, human TNF-α and mouse IL-1β were from Peprotech, HMW poly(I:C) and puromycin were from InvivoGen, and doxycycline was from Melford.
Torres A.A., Macilwee S.L., Rashid A., Cox S.E., Albarnaz J.D., Bonjardim C.A, & Smith G.L. (2022). The actin nucleator Spir-1 is a virus restriction factor that promotes innate immune signalling. PLoS Pathogens, 18(2), e1010277.
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9

Gene Silencing and IFNα Exposure in β Cells

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NLRC5 gene expression was silenced using two independent siRNAs [Thermo Fisher Scientific; siNL#1 (siRNA HSS130675): 5′-GGACACCUGGCAGUCUUUCAUUCAU-3′; siNL#2 (siRNA HSS130676): 5′-GCAGUUGGCAGAGUCUCUCGUUCUU-3′]. AllStars Negative Control siRNA (siCTL) (Qiagen) was used as a negative control; the siRNA control does not interfere with β cell gene expression, function, or viability (82 (link)). NOVA1 gene expression was silenced using an siRNA [Thermo Fisher Scientific; siNO1 (siRNA HSS143142): 5′-UUUGCAACUGAACAAUUGUCUGUCC-3′] (44 (link)). Cells were transfected using the Lipofectamine RNAiMAX lipid reagent (Invitrogen, Life Technologies) in Opti-MEM (Gibco, Thermo Fisher Scientific) reduced serum medium, according to the manufacturer’s instructions. EndoC-βH1 was transfected using 30 nM of each siRNA, overnight. Dispersed human islets and SC-derived β-like cells were transfected with 60 nM of each siRNA, also during an overnight incubation period. After transfection, EndoC-βH1, dispersed human islets, and SC-derived β-like cells were kept in culture for a 48-hour recovery period and subsequently exposed, or not, to human IFNα (2000 U/ml; PeproTech) for 8 or 24 hours. These conditions are based not only on previously published time dose-response experiments (8 (link)) but also on time course and dose response experiments presented in this article (fig. S4).
Szymczak F., Alvelos M.I., Marín-Cañas S., Castela Â., Demine S., Colli M.L., Op de Beeck A., Thomaidou S., Marselli L., Zaldumbide A., Marchetti P, & Eizirik D.L. (2022). Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic β cells. Science Advances, 8(37), eabn5732.
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10

Hepatocyte Cell Culture and Antibody Sourcing

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HepG2 and HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% inactivated fetal bovine serum, including penicillin (100 IU/mL) and streptomycin (100 mg/mL) under a 5% CO2 atmosphere at 37°C. PHH cells were pruchased from RILD (Research Institute for liver Diseases, Shanghai, China), HBx antibody (GS968942, Gilead Sciences) was kindly provided as a gift from Dr. Simon Fletcher. The expression constructs of TRIM5γ were generated by cloning the sequence of the coding region into a VR1012 expression vector. Anti-GAPDH antibody, anti-HA-tag, and GST-tag antibody were obtained from Proteintech. Human IFN-α was purchased from Peprotech (Jiangsu, China). DDB1, STAT1, and STAT3 antibodies were procured from Cell Signaling Technology (Danvers, MA, United States). Anti-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States), Antibodies against TRIM5γ and Smc6 were purchased from Abcam (Shanghai, China), Stattic was obtained from Sigma (Danvers, MA, United States).
Song H., Xu F., Pang X., Xiao Q., Wei Q., Lei B., Li X., Fan X, & Tan G. (2021). STAT3-Dependent Gene TRIM5γ Interacts With HBx Through a Zinc Binding Site on the BBox Domain. Frontiers in Microbiology, 12, 663534.
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