Real-time RT-PCR was performed as described previously [106 (link)]. MDMs were treated with 1,000 U/mL of IFN-α (Sigma) for 24 h of IFN-α in selected experiments. Alternatively, MDMs were transfected with siRNAs (see below). Total RNA was isolated using an RNeasy mini kit (QIAGEN), and cDNAs were synthesized using an oligo(dT)12-18 primer and M-MLV reverse transcriptase (Invitrogen). Then, real-time RT-PCR was performed with ApoE, MX2 or β-actin primer sets and SYBR Premix Ex Taq II (TaKaRa-Bio) using a LightCycler Nano (Roche) with 35 cycles. Primer sequences are as follows: 5’-AGCTGGTTCGAGCCCCTGGTG-3’ (forward) and 5’-TCAGTGATTGTCGCTGGGCAC-3’ (reverse) for ApoE, 5’-ATGTCTAAGGCCCACAAGCCT-3’ (forward) and 5’-TGGCACTGTGCCGAATGGCGG-3’ (reverse) for MX2, 5’-TGACGGGGTCACCCACACTG-3’ (forward) and 5’-AAGCTGTAGCCGCGCTCGGT-3’(reverse) for β-actin.
Ifn α
Manufactured by Merck Group
Sourced in United States, Germany
IFN-α is a type of interferon, a class of proteins produced by the body's cells in response to the presence of certain viruses, bacteria, or other foreign substances. IFN-α is primarily used in research and scientific applications to study the role of interferons in various biological processes.
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Lab products found in correlation
Ifn γ, by Merck Group (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Ifn γ, by Thermo Fisher Scientific (3 mentions)
Il 1β, by Merck Group (3 mentions)
Cd19 pe lt19, by Miltenyi Biotec (2 mentions)
Cd138 allophycocyanin b b4, by Miltenyi Biotec (2 mentions)
Cd38 af700, by BioLegend (2 mentions)
Cd20 efluor v450 2h7, by Thermo Fisher Scientific (2 mentions)
Cd27 af647 lt27, by Bio-Rad (2 mentions)
Cd19 percp cy5.5 sj225c1, by BD (2 mentions)
Cd24 fitc ml5, by BD (2 mentions)
Cd27 bv605, by BioLegend (2 mentions)
Cd3viogreen bw264 56, by Miltenyi Biotec (2 mentions)
Unconjugatedgoat anti irf4 m 17, by Santa Cruz Biotechnology (2 mentions)
Donkey anti goat igg af488, by Thermo Fisher Scientific (2 mentions)
Hybridomax hybridoma growth supplement, by Gentaur (2 mentions)
Mem amino acids solution, by Merck Group (2 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
Lipid mixture 1, by Merck Group (2 mentions)
67 protocols using ifn α
1
Real-time RT-PCR for ApoE and MX2 Expression
2
Minocycline Pretreatment Attenuates IFN-α-Induced Effects
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Recombinant human IFN-α was obtained from PeproTech Inc. (#300-02AB; Rocky Hill, NJ, USA) and was dissolved in artificial cerebrospinal fluid (ACSF; glucose, 5 mM; CaCl2, 1 mM; NaCl, 125 mM; MgCl2, 1 mM; NaHCO3, 27 mM; KCl, 0.5 mM; Na2SO4, 0.5 mM; NaH2PO4, 0.5 mM; and Na2HPO4, 1.2 mM). Rat serum albumin (1 mg) was added to 1 ml of 2 × 107 IU/ml IFN-α. The rats received bilateral infusions of ACSF (vehicle) or IFN-α at doses of 100, 200, or 400 IU/μl (1 μl/side).
Minocycline hydrochloride (#M9511; Sigma, St. Louis, MO, USA) was dissolved fresh in 0.9 % NaCl and administered intragastrically (i.g.) once daily at a dosage of 90 mg/kg rat body weight for 3 days prior to the IFN-α treatment. The dose was selected on the basis of previous studies showing the beneficial effects of this dosage in animal models of cerebral brain ischemia, multiple sclerosis, and Parkinson’s disease [29 (link)–33 (link)].
Minocycline hydrochloride (#M9511; Sigma, St. Louis, MO, USA) was dissolved fresh in 0.9 % NaCl and administered intragastrically (i.g.) once daily at a dosage of 90 mg/kg rat body weight for 3 days prior to the IFN-α treatment. The dose was selected on the basis of previous studies showing the beneficial effects of this dosage in animal models of cerebral brain ischemia, multiple sclerosis, and Parkinson’s disease [29 (link)–33 (link)].
3
Interferon alpha-2a stimulation of PBMCs
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Whole blood samples were collected from consenting healthy volunteers. PBMC were isolated using SepMate tubes (Stemcell Technologies) and Histopaque density medium (Sigma-Aldrich). Cells were cultured at 1-2 million cells/mL in RPMI (Gibco) supplemented with 10% autologous serum and Penicillin-Streptomycin-Glutamine (Gibco). Cells were treated with 1000 IU/mL recombinant human interferon alpha 2a (IFN-α; Sigma-Aldrich) for 3 hours unless otherwise indicated. Where applicable, 100 nM dexamethasone (DEX) (Sigma-Aldrich) was added after 1 hour of culture. For experiments including the Jak1/Tyk2 inhibitor tosylate salt (TS, Sigma-Aldrich), cells were treated with 0.25 or 1 µM TS for 1 hour prior to IFN-α addition. Studies were approved by the Monash Health human research ethics committee.
4
Cytokine Stimulation of Immune Cells
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PBMC or NK cells were cultured overnight before treating with purified IFN‐α (Sigma) or recombinant IFN‐α (Miltenyi Biotec), recombinant IL‐12 (R&D systems and Peprotech) or recombinant IL‐15 (Miltenyi Biotec) as described in the text and figure legends. For IFN‐I neutralization, a cocktail of anti‐human interferon α/β receptor chain 2 antibody (clone MMHAR‐2), anti‐human interferon‐α (sheep polyclonal) and anti‐human interferon‐β (sheep polyclonal; all from PBL Assay Science) or a control cocktail of mouse IgG2a (BioLegend) and sheep serum (Sigma) was used, as described previously [25 (link)].
5
Transcriptomic Profiling of IFNα-Treated Cells
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Total RNA was extracted, using the RNeasy Plus Mini Kit (QIAGEN), from MT4-LTR-GFP cells, control subclones, and CRISPR knockout subclones that were untreated or treated with 25 U/ml IFNα (Sigma) for 24 hr before harvest. cRNA was prepared and probed using Human HT12 Expression Beadchip (Illumina), containing ∼48,000 transcript probes, according to the manufacturer’s instructions.
6
Multiparametric Flow Cytometry of B Cells
7
Comprehensive Multicolor Flow Cytometry for Plasma Cell Analysis
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Abs used were CD19 PE (LT19; Miltenyi Biotec), CD138 allophycocyanin (B-B4; Miltenyi Biotec), CD38 PE-Cy7 (HB7; BD Biosciences), CD38 AF700 (HIT2; BioLegend), CD20 eFluor V450 (2H7; eBioscience); CD27 AF647 (LT27; AbD Serotec), CD27 FITC (M-T271), CD19 PerCP-Cy5.5 (SJ225C1; BD Biosciences), CD19 PE-Cy7 (SJ225C1; BD Biosciences), CD24 FITC (ML5; BD Biosciences), CD84 PE (CD84.1.21; BioLegend), CD38 PerCP-Cy5.5 (HIT2; BD Biosciences), CD95 BV421 (DX2; BioLegend), CD20 allophycocyanin-H7 (L27; BD Biosciences), CD27 BV605 (O323; BioLegend), CD3 VioGreen (BW264/56; Miltenyi Biotec), Ki67 FITC (B56; BD Biosciences), unconjugated goat anti-IRF4 (M-17; Santa Cruz Biotechnology), and donkey anti-goat IgG AF488 (polyclonal; Invitrogen). Controls were isotype-matched mouse mAbs. Annexin V FITC was from eBioscience, and 7-AAD was from BD Biosciences.
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)2 fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)2 fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).
8
Comprehensive Molecular Toolkit for Cellular Studies
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For regents, CHX (Sigma, St Louis, MO, USA), MG132 (MedChemExpress, Monmouth Junction, NJ, USA), 3-MA (MedChemExpress), CHQ (MedChemExpress), Bort (MedChemExpress), and IFNα (Sigma) were used to treat cells. For plasmids, ISG15 and 6PGL expression plasmids were bought from Origene (Beijing, China). LentiCRISPR v2 based constructs were used for knockout ISG15, UbCH8, HERC5, 6PGL, SMADs, TEADs. pGL4.21 vector was used to construct a 6PGL promoter-luciferase vector. YAPK280R-HA, YAPK321R-HA, YAPK497R-HA, 6PGLMut-P1, 6PGLMut-P2, 6PGLMut-P3, and 6PGLMut-P1+P2+P3 mutant plasmids were constructed using overlapping PCR. YAP, ATG5, PSMB5, and βTrCP knockout constructs, YAPWT-HA, YAPFLAG, SMAD2, TEAD4, RUNX2, TFCP2, P73, pUAS-Luc/TEAD-Gal4 plasmids were acquired from previous studies [15 (link), 31 (link), 43 (link), 49 (link), 50 (link)]. The primers are listed in Supplementary Table 1 .
9
Promoter Analysis of Human OAS3 Gene
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The promoter of human OAS3 (500 bp, Chr12:113375900–113376399) was cloned into the pGL3-Basic vector (Promega, E1751) upstream the luciferase gene at the BglII and HindIII restriction sites (pOAS3-luc), and into the pGL3-Promoter vector (Promega, E176; containing the SV40 promoter) downstream the luciferase gene at the BamHI and SalI restriction sites (pSV40-luc-pOAS3). Site-specific mutagenesis of the pOAS3 was done using the Q5 site-directed Mutagenesis Kit (NEB, E0554S) using a set of primers listed in Supplementary Data 7 . For cell transfection, 1 × 106 K562 cells were mixed with 1 μg of each construct and 200 ng of Renilla vector using the Neon Transfection System (Thermo Fisher Scientific; pulse voltage 1,450 V, pulse width 10, pulse number 3) and cultured in 2 mL in 12-well plates. After 18 h, half of the cell population was treated with 100 ng of IFNα (Sigma Aldrich, 50 ng/mL) for 6 h. Data were normalized to Renilla values and represented as the fold-change of relative light units over the wild-type pOAS3-luc or pSV40-luc-pOAS3 vector from non-stimulated K652 cells. Experiments were performed in triplicate.
10
Measuring IFNα in Infected Mice
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IFNα (Sigma) ELISAs were performed based on manufacturer’s instruction using serum and BAL fluid from uninfected and infected mice. In short, Triton X-100 and Rnase A were added to serum and BAL samples at final concentrations of 0.5% and 0.5 mg/ml, respectively, and incubated at room temperature (RT) at least 1 hour before use to reduce risk from any potential virus in serum and BAL. For serum IFNA levels, 20uL of serum and for BAL IFNA levels, 100uL of BAL fluid were used.
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