Supplement METHODS

Supplement METHODS

Cell culture. Cell line A549 was purchased from the ATCC, lung cancer cell line U1810 was characterized earlier [17, 18]. Both cell lines were cultured in DMEM, 4500 mg D–glucose/L, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 1mg/ml streptomycin (complete DMEM) in a 95% humidified atmosphere, in 5% CO2 at 37oC. Cells were passaged at a 1:10 ratio every 5 days following trypsinization with 0.05% trypsin–EDTA.

Isolation of stable clones of U1810 and A549 cells. Construction of siRNA interference plasmids and clones was described earlier [17, 18]. U1810 or A549 cells maintained in DMEM medium with 10% Fetal Calf Serum (FCS) on 24-well plate were transfected with mU6neo which expressed siRNA against PRXV, using Lipofectamine 2000 (Invitrogen). 0.8 µg of plasmid DNA or 1.6 µl of Lipofectamine were diluted in 50 µl of Opti-MEM, incubated for 5 min, then mixed and incubated for additional 20 min before addition to cells. After overnight incubation at 37oC in CO2 incubator, DMEM containing 10% FCS was added and incubation continued for 10h. Then cells were re-plated at 1/10 – 1/40 dilution onto the 6-well plates and selection with G418 (1 mg/ml) started on the next day. Individual clones growing on G418 were analyzed using Western blot with antibodies to PRXV. Two of the clones, which showed decreased expression of PRXV (as well as the mix of control clones obtained after transfection of A549 and U1810 cells with GFP siRNA expressing mU6neo plasmid), were further used in this study.

Temporary transfection of A549 and Calu-3 with plasmids carrying short (MAPIK-PRXV) or full length PRXV sequences. Construction of plasmids with full-length PRXV or short sequence was described in detail earlier [18]. Temporary transfection was done using Lipofectamine 2000 (Invitrogen). 0.8 µg of plasmid DNA or 1.6 µl of Lipofectamine were diluted in 50 µl of Opti-MEM, incubated for 5 min, then mixed and incubated for additional 20 min before addition to cells. After overnight incubation at 37oC in CO2 incubator, DMEM containing 10% FCS was added and incubation continued for 24-48h. Effectiveness of transfection was tested in a separate set of studies, where GFP-expressing plasmid was used under similar conditions. Quantification of the number of transfected cells demonstrated stable transfection levels of more than 80%. Overall effectiveness of transfection was further confirmed by Western blot analyses for PRXV in known number of cells.

Analysis of cell survival. For Trypan blue assay, cells were plated (6x104 cells per 5 cm2 plate) and 36 h later, medium was changed, and CSE or menadione was added for additional 24 or 48h incubation. Untreated cells were incubated in the same medium for similar time intervals. After washing with PBS, cells were stained for 1.5 min with 0.2% Trypan blue (Sigma, St. Louis, MI), and positive (dead) cells were counted in randomly selected three fields in binocular microscope (at least 300 cells per single view).

Western Blot Analyses. Western blots were performed for PRXV and reference protein (actin or GAPD). The epithelial cells were scraped off to a cold lysis buffer composed of 1% Triton X-100, 2 mM EDTA, 2 mM dithiothreitol, 0.25 mg/ml leupeptin, 0.25 mg/ml pepstatin A, 0.4 mg/ml aprotinin and 0.1 mM phenylmethylsulfonyl fluoride. In cell culture experiments, samples from 3 cell sheets were combined in 100 l of ice-cold buffer. Protein content of the lysate was measured by the BCA method (Pierce Endogen). Samples (20 g total protein) were separated on 10% SDS-polyacrylamide gels, and then separated proteins were transferred to PVDF membranes, where they were blocked overnight at 4˚C with 5% nonfat dry milk in TBST (10mM TrisCl, pH 8.0; 150 mM NaCl; 0.1% Tween 20). The blot was rinsed twice with TBST and incubated for 2 hours at room temperature with rabbit polyclonal PRXV (1:2000) antibody [16] in TBST containing 5% BSA. The membrane was washed for 15 min with TBST and incubated with goat anti-mouse IgG conjugated with horseradish peroxidase in 5% milk for 1 hour, and then washed three times with TBST and developed with enhanced chemiluminescence reagent (Amersham, Arlington, IL). Staining was quantified using Adobe Photoshop 7.0 Software.

Immunofluorescence. Slides with fixed cells were washed with PBS, postfixed with ethanol, washed twice, exposed to 0.2% Triton X-100 in PBS for 5 min and then washed with PBS twice again. Tissue sections were further blocked with 3% BSA/3% goat serum/0.1% Tween 20 in 4xSSC for 20 min. Appropriate primary antibody (in 2xSSC/1%BSA/0.1% Tween 20) was added for 1 hour at 37ºC, slides were washed 3 times with PBS and blocked with 3% goat serum/3% BSA/ 0.1% Tween 20 in 4xSSC for 20 min. FITC or Rhodamine - conjugated secondary antibody (in 2xSSC/1%BSA/0.1% Tween 20) (Molecular Probes, Eugene, OR), was added for 1 hour at 37ºC. Control samples were stained without primary antibody in parallel in all tissues. Sections were stained with PI (0.5 μg/ml) for 3 min, washed 3 times with PBS, mounted on glass slides in Fluoro-Guard antifade reagent (BioRad, Hercules, OR) and coverslips were applied. Laser confocal fluorescence microscopy (LSM 510, Zeiss, Germany) was performed at the UC Davis Department of Medicine Confocal Imaging Core Facility.

Assessment of epithelial permeability to albumin and tight junctional permeability in Calu-3 cells. Methods used to determine permeability of Calu-3 epithelial sheets to TexasRed-labeled albumin (Molecular Probes, Eugene, OR), and estimation of transepithelial electrical resistance are described in details in our recent publication [32]. Calu-3 cells form tight junctions in vitro, thus allowing to measure barrier functions of epithelium. Albumin was added to the apical side of epithelial layers and label concentration was measured at baso-lateral side 4-24 hours later. Transepithelial electrical resistance (measure of tight junctional ion permeability) was measured using EVON-METER (Precision Instruments, Sarasota, FL). Calu-3 cells were grown on 0.3 μm-pore Costar filters in DMEM with 10% Fetal Calf Serum (Gibco) for 6-12 days on air-liquid interface, until confluence was reached. Cells were transfected with IRES or PRXV-carrying plasmid 2 days prior to the experiment, similarly to as above-described method for A549 cells, effectiveness of transfection was confirmed by Western blot. Experiments were performed in 4-6 separate cell cultures and results averaged.

Treatment of cell cultures. Cigarette smoke extract was prepared as described earlier [30]. Sterile CSE was freshly prepared in PBS before each experiment and was used at concentrations 2-5% for the treatment of 2-48 hours, as specified in the Results. Hydrogen peroxide was used at 1-2 mM concentration, freshly prepared from 30% stock solution (Sigma). Menadione (Sigma, St. Louis, MO) was used at concentration 50 μM for the treatment duration 2-48 hours.