Saecs

HEp-2 and A549 cells were purchased from the ATCC (Manassas, VA), and were maintained in F12K and MEM medium respectively, containing 10% (v/v) FBS. SAECs, isolated from the normal human lung distal portion, were purchased from Lonza (Pittsburgh, PA), and were maintained according to the manufacturer’s protocol. RSV long strain was propagated in HEp-2 cells at 37 °C and purified by sucrose gradient as previously described.^{58 (link), 70} Viral titer was determined by immunostaining in HEp-2 cells using polyclonal biotin-conjugated goat anti-RSV antibody (7950–0104; Bio-Rad, Hercules, CA) and streptavidin peroxidase polymer (S2438; Sigma-Aldrich, St. Louis, MO) sequentially, as previously described.^{58 (link), 70} The polyclonal biotin-conjugated goat anti-RSV antibody was also used for Western blot to detect viral protein expression. The monoclonal antibody against β-actin was from Sigma (A1978). Primary antibodies against IRF-3(CST#4302), phospho-IRF3(Ser396)(#29047), p65(CST#4764), phospho-p65(Ser276)(CST#3037), and phospho-p65(Ser536)(CST# 3033) were purchased from Cell Signaling Technology (Denvers, MA), and goat anti-rabbit IgG-HRP (4050–05) was purchased from SouthernBiotech (Birmingham, AL).

A549 cells, a human alveolar type II like epithelial cell line (American Type Culture Collection, Manassas, VA) and small alveolar epithelial cells (SAECs)(Lonza Inc., San Diego, CA), normal human AECs derived from terminal bronchioli, were grown according to the manufacturer’s instructions. RSV infection in A549 cells were done in F12K medium containing 2% FBS. When SAECs were used for RSV infection, they were changed to basal medium, not supplemented with growth factors, 6 h before and throughout the length of the experiment. At 90 to 95% confluence, cell monolayers were infected with RSV at multiplicity of infection (MOI) of 3. An equivalent amount of 30% sucrose solution was added to uninfected A549 and SAECs, as a control.
For Anacardic acid (10 μM) (172050, EMD Millipore, MA) and Cerdulatinib (5 μM) (PRT062070, Selleckchem, TX) experiments, cells were pretreated with the compounds for 1 h and then infected in their presence for the duration of the experiment. Equal amounts of diluent were added to cells uninfected and infected as control. Total number of cells and cell viability, following various treatments, were measured by trypan blue exclusion. There was no significant change in cell viability with all compounds tested. There was no effect of Anacardic acid on viral replication, while there was a trend in increased viral replication in cells treated with Cerdulatinib.

Human small airway epithelial cells (SAECs) were
purchased from Lonza, Allendale, NJ (LOT: 000470903). Primary mouse AECIIs were
isolated from the lungs of unchallenged mice. Mouse lung epithelial cell line
(MLE12) was purchased from ATCC (CRL-2110). Cells were seeded into 96-well
plates (40.000/well), exposed to bleomycin (15 mU/ml) or PBS for 4 hours and
then treated with T3 (15 ng/ml) or vehicle control for 8 hours.
Immunofluorescence staining was performed using Mito-Tracker (Thermo Fisher
Scientific, Waltham, MA), a cationic dye that stains active mitochondria and
PPARGC1A (ab54481, Abcam, Cambridge, UK), according to manufacturer’s
instructions. Detection of apoptotic cells was performed with TUNEL-assay using
the in situ Cell Death Detection Kit, Fluorescein (Roche, Indianapolis, IA, USA,
Catalog No 11684795910). DAPI staining was used to determine the number of
nuclei and to assess gross cell morphology. The human lung adenocarcinoma cell
line A549 (ATCC^® CCL-185™, ATCC, Manassas, VA),
negatively tested for mycoplasma, was cultured in DMEM supplemented with
10% fetal bovine serum. Cells were pre-incubated with either dronedarone
10 μM diluted in 0.04% DMSO or vehicle control (0.04%
DMSO) for 24 hours and then treated with T3 (15 ng/ml) or vehicle (normal saline
0.9%) for 24 hrs.