*2.1. Preparation of CSE*

CSE was obtained from 3R4F research cigarettes (University of Kentucky, Lexington, KY, USA), with each cigarette containing 0.60 mg of nicotine and 8.0 mg of tar, for use in all experiments. CSE was prepared by bubbling smoke from cigarettes into 15 mL of serum-free Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island, NY, USA) at a rate of 1 cigarette/min using a modification of the method developed by Carp and Janoff [16]. The CSE solution is defined as 100% CSE and diluted with DMEM in the following experiments. CSE was standardized by measuring of the absorbance at 320 nm confirmed that the prepared CSE was reproducible, and various CSE preparations showed few differences. Freshly prepared CSE was used immediately in all the experiments.

### *2.2. Patients and Tissue Collection*

Sinus tissue explants were collected during surgery from patients with blowout fracture (*n* = 6) at the Department of Otorhinolaryngology at Korea University Guro Hospital, Korea. All patients had no history of smoking, allergies, asthma, or aspirin sensitivity, and they were not treated with any antibiotics or oral or topical steroids for at least 4 weeks before surgery. Written consent was obtained from all patients, and the study was approved by the Ethics Committee of the Faculty of Medicine, Korea University, Korea.

### *2.3. Nasal Fibroblast Cultures*

To obtain nasal fibroblasts, cells were cultured in DMEM with 10% heat-inactivated fetal bovine serum (FBS), 1% (*v*/*v*) 10,000 U/mL penicillin, and 10,000 µg/mL streptomycin (Invitrogen) in a humidified incubator under 5% CO<sup>2</sup> at 37 ◦C. Experiments were performed using cells at 80% confluence. Before treatment of agents, the cells were starved in serum-free media for 12 h. The purity of obtained human nasal fibroblasts was confirmed microscopically based on the characteristic spindle-like cell phenotype. Approximately 95% of cells in cultured nasal fibroblasts were positive for vimentin and Thy-1, which were used as fibroblast markers, and negative for E-cadherin, which was used as an epithelial cell marker. The nasal fibroblasts were cultured for four passages [17].

### *2.4. Real-Time PCR*

The nasal fibroblasts were incubated with CSE for 12 h after pre-treatment of ROS scavengers (NAC, ebselen and DPI) PI3K/Akt inhibitor (LY294002) and NF-κB inhibitor (BAY11-7082) for 1 h. RNAs were extracted from nasal fibroblasts using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The total amount of RNA was determined using NanoDrop 2000 (Thermo Fisher Scientific Inc., Wilmington, DE, USA). Synthesis of cDNA was performed with 1 µg of total RNA using the Maxime RT PreMix cDNA Kit (iNtRON Biotechnology, Sungnam, Korea). The expression levels of mRNAs were analyzed using Quantstudio3 (Applied Biosystems, Foster City, CA, USA) and Power SYBR Green PCR Master Mix (Applied Biosystems). Real-time PCR analysis was performed to evaluate the expression levels of *MMP-2, MMP-9, TIMP-1,* and *TMIP-2* and that of *GAPDH*, which was used as a housekeeping gene. The primer sequences are shown in Table 1. The results were normalized by *GAPDH* mRNA expression and are shown as the fold ratio over the expression of the control group.


**Table 1.** Sequences of real-time PCR oligonucleotide primers.

#### *2.5. Gelatin Zymography*

Nasal fibroblasts were exposed to CSE for 72 h after pretreatment with ROS scavengers, PI3K/Akt inhibitor and NF-κB inhibitor for 1 h. Aliquots of fibroblast-conditioned medium (10 µL) were analyzed using gelatin zymography for MMP-2 and MMP-9 in 1 mg/mL gelatin-10% polyacrylamide gels. Following electrophoresis, the gels were washed twice with 2.5% Triton X-100 for 30 min while shaking to remove sodium dodecyl sulfate and renature MMP-2 and MMP-9 in the gels. Renatured gels were incubated in developing buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35 overnight at 37 ◦C. Gels were stained with 0.25% Coomassie brilliant blue G-250 (50% methanol, 10% acetic acid) and destained using destaining solution (50% methanol, 10% acetic acid). Proteinase activity was observed as cleared (unstained) regions on the gels. Finally, the gels were dried for 2 h using a gel dryer (Bio-Rad, Hercules, CA, USA).

#### *2.6. Enzyme-Linked Immunosorbent Assay (ELISA)*

MMP-2, MMP-9, TIMP-1, and TMIP-2 concentrations in the culture media were determined using ELISA (R&D systems, Minneapolis, MN, USA). Nasal fibroblasts were exposed to CSE for 72 h after pretreatment with ROS scavengers, PI3K/Akt inhibitor and NF-κB inhibitor for 1 h. Standards and samples were added and incubated at room temperature for 2 h. After 3 washes, MMP-2, MMP-9, TIMP-1, or TMIP-2 conjugate was added to the wells for 2 h at room temperature. The reaction was stopped with a stop solution, and the product was quantified at 450 nm using a microplate reader (Bio-Rad).

#### *2.7. Western Blotting Analysis*

Nasal fibroblasts were treated with CSE for 72 h. A total of 5 <sup>×</sup> <sup>10</sup><sup>5</sup> fibroblasts were lysed in PRO-PREPTM protein extraction solution (iNtRON Biotechnology) and stored overnight at <sup>−</sup><sup>20</sup> ◦C. Cell debris was removed from the lysates by centrifugation at 13,000× *g* for 30 min at 4 ◦C. Total protein concentration was determined using the Bradford assay (Bio-Rad). An equal quantity of protein from samples (30 µg) were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to 0.45 µm polyvinyl difluoride membranes, (Millipore Inc., Billerica, MA, USA), and analyzed separately. Membranes were blocked with 5% skim milk at room temperature for 60 min, rinsed three times with Tris-buffered saline containing Tween-20, and treated with the following primary antibodies: polyclonal p-PI3K (1:1000, #4228, Cell Signaling Technology, Danvers, MA, USA), total-PI3K (1:1000, #4292, Cell Signaling Technology), p-Akt (1:1000, #9271, Cell Signaling Technology), total-Akt (1:1000, #9272, Cell Signaling Technology), p-p65 (1:1000, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), total-p65 (1:1000, sc-8008, Santa Cruz Biotechnology Inc.) and β-actin (1:10000, sc-47778, Santa Cruz Biotechnology Inc.). Bands were visualized using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence system (Pierce, Rockford, IL, USA).

### *2.8. Immunofluorescent Staining*

Nasal fibroblasts were placed onto coverslips and treated with CSE for 72 h. Fibroblasts were fixed with 4% paraformaldehyde and then permeated with 0.2% Triton X-100 in 1% FBS for 10 min at room temperature. After treating coverslips with 5% BSA to block for 1 h at room temperature, fibroblasts were incubated overnight at 4 ◦C with anti-MMP-2, anti-MMP-9, anti-TIMP-1, or anti-TIMP-2 antibodies (Santa Cruz Biotechnology, Inc. CA, USA). Goat anti-mouse Alexa 488 (Invitrogen) secondary antibody was also added to fibroblasts and incubated. Lastly, 40 -6-diamidino-2-phenylindole (DAPI) was applied for counterstaining. The stained normal fibroblasts were subsequently maintained on a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany).

#### *2.9. Measurement of ROS Produced*

Intracellular ROS production was determined using a fluorescent probe, 2, 7-dichlorodihydrofluorescein diacetate (Molecular Probes, Inc., Eugene, OR, USA). Cells were pretreated with N-acetyl-L-cysteine (NAC, 5 mM), ebselen (10 µM), or diphenyleneiodonium (DPI, 2 µM) for 1 h and then stimulated with CSE for 12 h. After that, CSE-stimulated cells were suspended in serum-free culture medium with H2DCF-DA (10 µM) for 30 min. Cellular fluorescence was measured using fluorescence microscopy (IX71; Olympus, Life Science Solutions, Tokyo, Japan) to observe ROS production. The H2O<sup>2</sup> production was measured using an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific Inc.), according to the manufacturer's instructions. Nasal fibroblasts were pretreated with NAC, ebselen, or DPI. Prior to CSE stimulation, the cell lysate (50 µL) was treated with a working solution of 100 µM Amplex Red reagent (Thermo Fisher Scientific Inc.) and 0.2 U/mL horseradish peroxidase. After incubation for 30 min at 37 ◦C, fluorescence was measured at 560 nm on a microplate reader (Bio-Rad).

#### *2.10. Statistical Data Analysis*

For all outcomes, data were obtained in triplicate from at least three separate experiments. Data are shown as mean ± SEM of three different experiments in triplicate. All significant differences between controls and examined samples were analyzed by one-way analysis of variance followed by Tukey's test (GraphPad Prism version 5; GraphPad Software, San Diego, CA, USA). Significance was considered at a 95% confidence level. P-values below 0.05 were considered statistically significant.

#### **3. Results**
