*2.3. Animal Experiments*

Six week-old male BALB/c mice (Hallym University Breeding Center for Laboratory Animals) were used in this study. Female mice were excluded because of concerns that female hormone cycles would affect experiments. Mice were kept on a 12 h light/12 h dark cycle at 23 ± 1 ◦C with 50% ± 10% relative humidity under specific pathogen-free circumstances, fed a non-purified diet, and provided with water ad libitum at the animal facility of Hallym University. The present study was approved by the Hallym University Institutional Review Board and Committee on Animal Experimentation (Hallym 2017-56). This study was conducted in compliance with the University's Guidelines for the Care and Use of Laboratory Animals.

Mice were acclimatized for 1 week before beginning the experiments. All mice were distributed among five subgroups (*n* = 9–10 for each subgroup).

Passive smoking models: Mice receiving the smoke challenge were further divided into four subgroups. One subgroup (no CS) did not receive the smoke challenge. YE solution (containing 25–100 mg/kg BW) was orally administrated to mice 1 h via oral gavage once a day (5 days/week) for 8 weeks before. Subsequently, mice were exposed to smoke of research cigarettes (11 mg tar and 0.7 mg nicotine/cigarette) for 30 min in a specially designed chamber once a day for 8 weeks. Research cigarettes (3R4F, 11 mg tar and 0.7 mg nicotine per cigarette) were obtained from the University of Kentucky (Lexington, KY, USA).

Mouse asthma model: Mice receiving the OVA challenge were further divided into five subgroups. One subgroup (no OVA) did not receive the OVA challenge. Mice were sensitized with 20 μg OVA dissolved in 30 μL phosphate buffered saline (PBS) with 50 μL Imject Alum (Thermo Fisher Scientific, Rockford, IL, USA) through subcutaneous injections on the days 0 and 14. Subsequently, 0.1 mL YE solution (containing 25–100 mg/kg BW) was administered via oral gavage to OVA-sensitized mice 1 h before challenge. On the days of 28−30, 5% OVA inhalation to mice was carried out for 20 min in a plastic chamber linked to an ultrasonic nebulizer (Clenny2 Aerosol, Medel, Italy). Control mice were sensitized and challenged with PBS as the OVA vehicle. All mice were sacrificed 24 h after the latest provocation (day 30).

All the mice were killed with an anesthetic dose of 0.3 g/kg avertin and 8 μg/kg *tert*-amyl alcohol. The trachea was cannulated, and both lungs and airways were rinsed in 1 ml PBS for the collection of bronchoalveolar lavage fluid (BALF). The numbers of inflammatory cells including neutrophils and eosinophils in BALF were determined using a Hemavet HV950 Multispecies Hematologic Analyzer (Drew Scientific, Oxford, CT, USA). The right lungs were collected, frozen in liquid nitrogen, and kept at −80 ◦C until used for Western blotting. Left lungs were preserved and fixed in 4% paraformaldehyde and then used for immunohistochemical analyses.

#### *2.4. Preparation of CSE for Cell Culture*

The research cigarettes were consecutively smoked through an experimental apparatus with a constant airflow driven by an air vacuum pump. The collected CS was bubbled in 10 mL PBS. The resulting smoke suspension was filtered through a 0.22 μm pore filter in order to eliminate bacteria and large particles. The filtrates referred to a 100% CSE.

#### *2.5. A549 Cell Culture and Viability*

Human alveolar basal epithelial cells A549 cells were provided by the American Type Culture Collection (Manassas, VA, USA). A549 cells were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. A549 cells were sustained in 90–95% confluence at 37 ◦C in an atmosphere of 5% CO2. A549 cells were treated with 10–100 μg/mL YE and then stimulated with 2 μg/mL LPS or 5% CSE for up to 24 h to induce expression of target gene proteins.

The cytotoxicity of YE was determined using 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT, Duchefa Biochemie, Haarlem, Netherlands) after culture of A549 cells. These cells were incubated in a fresh medium containing 1 mg/mL MTT for 3 h at 37 ◦C. The purple formazan product was dissolved in 0.5 mL isopropanol with gentle shaking. Absorbance of formazan was measured at λ = 570 nm using a microplate reader (Bio-Rad Model 550, Hercules, CA, USA).

#### *2.6. Staining with Hematoxylin and Eosin (H&E)*

For the histological analyses of airways, small airway and alveolar specimens provided at the end of the experiments were fixed in 10% paraformaldehyde. The para ffin-embedded specimens were sectioned at 5 μm thickness, depara ffinized and stained with hematoxylin and eosin (H&E) stain for 2 min, and quickly dehydrated in 95% absolute alcohol. The H&E-stained tissue sections were observed using an optical microscope Axioimager system equipped for fluorescence illumination (Zeiss, Gottingen, Germany). Five images were taken from each tissue section.
