Stattic

Lipopolysaccharide from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich (St. Louis, MO, United States). Recombinant mouse IFN-γ protein was purchased from R&D systems. The bacterially expressed recombinant mouse C8G protein was prepared as described previously (Lee et al., 2008 (link)). Briefly, the recombinant mouse C8G protein without the signal peptide was expressed in E. coli (strain BL21) as a glutathione S-transferase (GST) fusion protein. The protein was purified using glutathione Sepharose 4B beads (GE Healthcare, Chicago, IL, United States). GST was removed by thrombin digestion. C8G was purified and concentrated. SDS-PAGE with Coomassie Brilliant Blue R250 staining showed that the purity of C8G was over 95%. JTE013, S1P, STATTIC, and BAY 11-7082 were purchased from Cayman Chemical (Ann Arbor, MI, United States). CYM-5478 was purchased from Aobious Inc. (Gloucester, MA, United States).

The putative human ACVRL1 promoter sequence (GeneBank: NC_000012.12, position 51906383 to 51907627) was amplified by the forward primer; 5ʹ- GGGGGTACCATAACCAGGAGGCTAGG-3ʹ and the reverse primer; 5ʹ-TTTAAGCTTCGCGGCCGCAGTTG-3ʹ. The obtained fragment was then subcloned into pGL3-basic vector (Promega) at the KpnI and HindIII sites^{29 (link)}. The construct was verified by restriction digestion and DNA sequencing. The pGL3-basic vector containing the putative ACVRL1 promoter region and pNL1.1.TK [Nluc/TK] as a control vector were co-transfected by using ScreenFect A Plus (Wako) according to the manufacturer’s protocol. The promoter activity of ACVRL1 was determined by using Dual-Glo Luciferase Assay System (Promega). The cells were incubated with ruxolitinib (Novartis Pharmaceuticals) or stattic (Cayman Chemical) for 24 h prior to the luciferase assay. Each experiment was performed in duplicate.

It is well known that poly(I:C) stimulates innate immunity such as TLR3.³³ Thus, it was used for surrogate viral RNA such as rhinovirus.¹³ We also used CpG oligonucleotides (CpG–ODN), TLR9 ligand replacement of viral DNA. Both nucleic acid compounds were purchased from Novus Biologicals. IL‐13 and IL‐4 were purchased from PeproTech. IL‐33 was purchased from Wako Pure Chemical. IL‐37 and CC16 (Clara cell secretory protein; a marker of bronchial lung epithelial cells) were purchased from ProSpec and R&D Systems, respectively. BAY 11‐7082 (an inhibitor of nuclear factor kappa light chain (NF‐κB) enhancer of activated B cells was purchased from InvivoGen. Ruxolitinib, stattic (an inhibitor of signal transducer and activator of transcription 3³⁴), and LY294002 (a phosphatidylinositol kinase‐3 inhibitor) were purchased from Cayman Chemical. The chemotherapy agent, fludarabine, was purchased from Wako Pure Chemical. The corticosteroid, FP, was purchased from Sigma. A type I interferon (IFNs) neutralizing antibody mixture was purchased from PBL Assay Science. Small interfering RNAs (siRNAs) for TLR3, interferon regulatory factor (IRF) 3, RelA (a NF‐κB subunit), JAK1, STAT6, extracellular signal‐regulated kinase (ERK) 1, ERK2, Stealth RNAi siRNA Negative Control Med GC Duplex #2, and Lipofectamine RNA iMAX Reagent were purchased from Invitrogen.