Subjects and reagents
Normal human bronchial epithelial (NHBE) cells (Cambrex Bio Science, Walkersville, MD) were purchased. Normal immortalized (BEAS-2B and 1799), transformed (1198) and tumorigenic (1170-I) HBE cells were obtained as kind gifts from Dr. Curtis Harris (National Institutes of Health, Bethesda, MD) 
and Dr. Andres Klein-Szanto (Fox Chase Cancer Center, Philadelphia, PA) 
. NHBE cells were grown in bovine epithelial growth media with SingleQuots supplements and growth factors (bovine pituitary extract, hydrocortisone, human epithelial growth factor, epinephrine, insulin, triiodothyronine, transferrin, gentamicin, amphotericin-B, retinoic acid, and bovine serum albumin-fatty acid free). The BEAS-2B and 1799 cells were grown in keratinocyte serum-free media (Gibco BRL, Eggstein, Germany) supplemented with EGF and pituitary extract. The 1198 and 1170-I cells were cultured in the same medium used in BEAS-2B cells, except that 3% FBS was added to the medium. The five HBE cell characteristics are summarized in . A549 cell line (American Type Culture Collection, Manassas, VA) was used as a positive control of malignant stage.
Characteristics of Five Human Brochial Epithelial Cell Lines.
Lung tumors and matched normal lung tissue samples were obtained from 12 lung cancer patients (Korea Lung Tissue Bank of Korea University Guro Hospital, Seoul, Korea). The study protocol was approved by the Institutional Review Board of Korea University Hospital and all patients provided written informed consent.
LY294002, RO31-8220 and Gö6976 (Calbiochem-Novabiochem, San Diego, CA) were prepared in dimethyl sulfoxide (DMSO). Nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma-Aldrich, St. Louis, MI) were prepared in DMSO or ethanol. Recombinant Wnt5a (R&D Systems Inc., Minneapolis, MN) was reconstituted in sterile phosphate buffered saline (PBS) containing 0.1% bovine serum albumin.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA). Briefly, 1 µg of total RNA was reverse transcribed into cDNA and amplified by PCR. The primers and PCR reaction conditions for Wnt5a have been described previously 
. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a positive amplification control. PCR products were visualized on 2% agarose gels with ethidium bromide. The PCR results were scanned and mean intensity ratio of Wnt5a/GAPDH analyzed using ImageJ software (NIH Image, National Institutes of Health, Bethesda, MD; online at: http://rsb.info.nih.gov/ij/
Quantitative real-time PCR analysis
Quantitative real-time PCR assay was performed using iQ SYBR Green Supermix (Bio-Rad, Hercuis, CA) according to the manufacturer's instructions. Briefly, 1 ul of reversely transcribed cDNA was used for the PCR reactions with the Wnt5a specific forward primers (CAA CTG GCA GGA CTT TCT CA) and reverse primers (CAT TCT TTG ATG CCT GTC TTC G). The PCR reaction was carried out as follows: initial denaturation at 95°C for 5 min followed by: 40 cycles of denaturation at 95°C for 30 s; annealing at 58°C for 30 s; and extension at 72°C for 1 min. The threshold cycle (CT) was analyzed using the PCR apparatus procedure and the relative copy number ratio of Wnt5a (ΔCT) and GAPDH (ΔCT) to determine the mRNA expression levels of Wnt5a. All PCRs were carried out in duplicates.
Small interfering RNA (siRNA) transfection
The siRNA duplexes for Wnt5a (D-003939-1) were obtained in a siGENOME SMART pool (Dharmacon Research Inc., Lafayette, CO). The siRNA for PKCα and β were purchased (Santa Cruz Biotechnology, Santa Cruz, CA). siRNAs were transfected into the 1799 cell line using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) for 48 h. Gene knockdown was verified by Western blotting for Wnt5a, PKCα and β. Control cells were treated with scrambled duplex.
Western blot analysis
Cells were lysed and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and resolved proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were then blocked and incubated with specific monoclonal antibodies; Wnt5a (R&D Systems, INC, Minneapolis, MN). Isoforms of phospho-PKC (phospho-PKC antibody sampler kit #9921, cell Signaling, Danvers, MA), phospho-PKC α/βII, phospho-PKC (pan), phospho-Akt, Akt and poly ADP-ribose polymerase (PARP; Cell Signaling, Danvers, MA), Bax and Bcl-2 (Santa Cruz Biotechnology), and β-actin (Sigma Chemicals, St Louis, MO).
Cell viability analysis
The cell viabilities were determined using trypan blue dye exclusion assay. Proportions of dead cells were determined using a hemocytometer.
Apoptosis was assessed by a flow cytometry-based terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) assay using an APO-bromodeoxyuridine (APO-BrdUrd) staining kit (Phoenix Flow Systems, San Diego, CA) according to the manufacturer's instructions.
Preparation of cigarette smoke condensate (CSC)
CSC was prepared as previous described. Briefly, one cigarette (Research Grade Cigarette, University of Kentucky) was combusted with a Variable Speed Pump (Fisher Scientific, Pittsburgh, PA). The smoke was bubbled through 25 ml keratinocyte-serum free media. The resulting suspension was filtered by a 0.22-µm pore filter. This solution was considered to be 100% CSC and diluted with keratinocyte serum-free media fresh before experiments.
Colony formation assay
After the treatment of 50% CSC every 48 h for 8 days, the survived cells were further incubated for 2 weeks and used for selecting the cells expressing high or low levels of Wnt5a, and then were measured the levels of Wnt5a expression in the cells. After confirming the expression status of Wnt5a, the cells (1×103) with/without the treatment of PKC inhibitor (2.5 µM Gö6o76) were plated on 6-well plates and cultured for 2 weeks. The cell images of colony formation were taken under the gel doc image analyzer (Bio-Rad, Hercuis, CA). The number and size of the stained colony was measured using ImageJ software (NIH Image, National Institutes of Health, Bethesda, MD).
All statistical analysis was conducted by the Student's t test. Analyses were carried out using SPSS (ver. 10.1; SPSS Inc., Chicago, IL, USA). A P value of <0.05 was considered significant.