LIFR protein, human

Calu-3 and MDCK cells (ATCC) were maintained in minimum essential media supplemented with 10% fetal bovine serum and Dulbecco's modified Eagle's media with 5% fetal bovine serum, respectively [26] . Human pBECs were obtained from healthy individuals by endobronchial brushing during fibre-optic bronchoscopy [67] (link). Subjects had no history of smoking or lung disease and had normal lung function. All subjects gave written consent. pBEC were cultured as described [26] , [35] (link). For differentiation of pBECs, cells were grown on transwells (Corning) at air-liquid interface, with basolateral media changed every second day at 37°C/5% CO₂[68] (link).
H3N2 and H5N1 were diluted in the appropriate serum free media and added to cells at multiplicity of infection (MOI) of 5 and of 0.005, respectively. After 1 h of incubation, the inocula were removed and replaced with serum-free media. Cells were treated with exogenous polyinosinic∶polycytidylic acid (Poly I:C, 100 µg/ml, Sigma-Aldrich), a known agonist of RIG-I and IFN responses, as a positive control [36] (link), [69] (link). Cycloheximide (100 ug/ml, Sigma-Aldrich) was used to inhibit protein synthesis by pre-treatment of cells (30 min, 37°C, 5% CO₂) and was added to media after virus inoculation. Caspases inhibitor Z-DEVD-fmk (Calbiochem, USA) of 50 µM was used to pre-treat the cells for 3 h before infection and was added after 1 hr virus inoculation. In experiments involving the blockade of IFNAR2, cells were incubated with 1 µg/ml of mouse monoclonal neutralizing antibody to IFNAR2 (CD118, PBL Laboratories) for 1 h prior to virus inoculation.

8-week old female mice in diestrus were perfused with ice cold PBS, brains rapidly removed and flash frozen in isopentane on dry ice. Coronal brain sections of 500 μm were obtained using vibratome, hypothalamus dissected, RNA isolated using the RNAqueous^®-Micro Kit (Ambion) and quantified using Nanodrop. Gene expression in 50 ng RNA per sample was analyzed using the Nanostring instrument as described before (54 (link)), according to manufacturer’s instruction, with the nCounter Mouse Neuroinflammation Panel (770 genes, gene list available at the manufacturer’s website). The panel was customized with an addition of 30 custom probes for: Gnrh1, Kiss1, Kiss1r, Pdyn, Tac2, Tac3r, Agrp, Npy, Pomc, Cart, Mc3r, Mc4r, Hcrt, Ghrh, Crh, Trh, Oxt, Avp, Prl, Prlr, Adcyap1, Slc6a3, Slc32a1, Slc17a6, Th, Lif, Lifr, Gabra5, Gabra1, Gabrg2. Only samples with an RNA integrity number RIN over 7 were used after passing QC, with no imaging, binding, positive control, or CodeSet content normalization flags. Data analysis was performed using nSolver Analysis Software 4.0, including nCounter Advanced Analysis (version 2.0.115). Genes with the expression lower than the limit of detection after background subtraction, and compared to negative controls included in the panel, were excluded. Seven housekeeping control genes that are included in the panel, were used for normalization. A heatmap of differentially expressed genes (DEG) was created using Heatmapper software from University of Alberta (Edmonton, Canada (55 (link));). Results are plotted in the Volcano plot as log fold change vs. log p-value, and genes with changes higher than 20% were indicated with colors in the figures: red indicates genes higher in KO compared to WT, while green indicates genes that are higher in the WT compared to KO. Genes with significant changes in expression are indicated above the dashed line. Gene ontology (GO) enrichment analysis of the DEG genes was performed using the ShinyGo 0.76.3 platform (South Dakota State University (56 (link))). False discovery rate (FDR) cutoff was 0.05, with the pathway minimum set to 10. Data is deposited in GEO repository (accession number GSE222723).

Whole cell lysates were produced by rinsing cells grown on culture plates twice with PBS, prior to scraping the cells into a small volume of PBS. Cells were pelleted by centrifugation at 300 × G in microcentrifuge tubes, and an appropriate amount of RIPA lysis buffer (50 mM Tris-Cl, pH 7.6, 1% NP-40, 12 mM sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 165 mM sodium chloride) was added. The cells were kept on ice with occasional mixing for ~45 min and then centrifuged at 16,000 × G at 4°C for ten minutes. The supernatants were then transferred into fresh tubes and stored at −20°C. Protein concentrations were measured using a Pierce Micro BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were denatured by adding an appropriate volume of Laemmli sample buffer (62.5 mM Tris-Cl pH 6.8, 1% SDS, 10% glycerol, 50 mM dithiothreitol (DTT), 0.001% bromophenol blue), followed by incubation at 95°C for 5 min. Denatured samples were resolved by electrophoresis through polyacrylamide gels ranging from 7.5% to 12.5% and electrotransferred onto polyvinylidene fluoride (PVDF) membranes at a constant current for 16–20 hours. Membranes were incubated in blocking buffer (5% skim milk in Tris-buffered saline with 0.01% Tween-20, TBST) for one hour at room temperature (RT), and then incubated for 2–24 hours at 4°C in blocking buffer with the addition of a primary antibody to detect the protein of interest. The following primary antibodies were used: LIFR (Santa Cruz #sc -515,337), FAM3C (Sigma #AV44904), pSTAT3 (Cell Signaling #9145), STAT3 (Cell Signaling #9139), GAPDH (Santa Cruz #sc -32,233), β-Actin (Santa Cruz #sc -47,778), TWIST1 (Cell Signaling #90445), and HSP90 (Santa Cruz #sc -13,119). After primary antibody incubation, the membranes were rinsed 3 X 10–15 minutes in TBST and then incubated in blocking buffer with the addition of the appropriate secondary antibody for 0.5–1.5 hours at RT. The following horseradish peroxidase (HRP)-conjugated secondary antibodies were used: goat anti-mouse IgG (Thermo Fisher #31430; 1:10,000) and goat anti-rabbit IgG (Thermo Fisher #31460; 1:10,000). After incubation with the secondary antibody, the membranes were washed again as described above. Bands were detected by adding 1–2 mL of HRP substrate (EMD Millipore) directly onto the membrane. Images were acquired using ChemiDoc MP (Bio-Rad) and processed using Image Lab software (Bio-Rad).

A DNA sequence corresponding to the mouse LIFR regulatory region (Table S3) was amplified from genomic DNA extracts and ligated into the pGL3-Basic vector (Promega). Approximately 100 K cells in 12-well culture plates were co-transfected with ~1 µg pGL3 and ~ 500 ng pNL3.1 (Promega) and allowed to grow for 24 h. The cells were then trypsinized and resuspended in DMEM containing 10% serum, centrifuged at 300 × G and washed 2X with PBS with repeated centrifugation. Cells were then resuspended in 200 µL of PBS and distributed into 96-well opaque white assay plates (triplicate wells at 60 µL). Luminescence was measured using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol using a Molecular Devices Spectramax iD5 Multi-Mode Microplate Reader. The relative normalized reporter signal was calculated by first subtracting “background signal” firefly luminescence (pGL3) acquired from pGL3-Basic “empty vector” control transfections from each experimental transfection, then dividing the remaining firefly luminescence values by the Nano-Luc luminescence (pNL3.1) values generated by the same well to determine the normalized reporter signal per well. Finally, the mean normalized reporter signal was determined from triplicate wells and compared between experimental groups. For experiments using the STAT3 CIE reporter, the pGL3 vector was replaced with the pGL4.47 vector (Promega). For experiments using STAT3 inhibition, the cells were seeded in DMEM and allowed to grow overnight. Immediately prior to transfection, the culture medium was replaced with a medium containing STAT3-IN-1 (10 µM final concentration) or an equal volume of DMSO.