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  • br Western blot analysis br After being subjected to


    2.7. Western blot analysis
    After being subjected to the indicated treatments, cells were lysed and protein concentration determined in the cell lysates as described elsewhere [20,24]. Proteins were resolved by 10% SDS/PAGE gel electrophoresis, transferred to a PVDF membrane (Millipore) and ana-lyzed by immunoblot after overnight incubation of the blots at 4 °C with the following primary PFK-158 [anti-α7 (1:500 dilution) or anti-β-catenin, anti-vimentin or anti-fibronectin (at 1:1000 dilution)]. The anti-β-actin antibody (1:1000) was incubated for 1 h at room tem-perature. Proteins were detected by blot incubation (1 h) with the ap-propriate HRP-conjugated secondary antibody (1:5000, Santa Cruz Biotechnologies, CA, USA). The resulting bands were detected using the ECL Select Western Blotting Detection Reagent (Amersham, GE Healthcare, UK).
    2.8. Mice model and in vivo tumor xenograft assays
    This study was approved by the Animal Care and Use Committee of the Universidad Autonoma de Madrid (UAM) in accordance with the EU Directive 2010/63/EU for animal experiments. Animals were housed under pathogen-free conditions in the Animal Resource Facilities of our institution (Instituto de Investigaciones Biomédicas Alberto Sols-CSIC-UAM). Five- to six-week-old female NU-Foxn1nu athymic mice were purchased from Charles River Laboratories (Wilmington, MA, USA). A suspension of wild-type A549 cells or of A549dupα7 cells (2 × 106 cells/ mouse) was subcutaneously injected into the left flank of each mouse. Ten days later, each group of mice inoculated with one or the other cell variant was randomly distributed into two subgroups (5–6 mice/  Lung Cancer 128 (2019) 134–144
    subgroup) according to whether they were going to receive nicotine or not in drinking water containing saccharin (2%). Mice were monitored once every three days to evaluate the growth rate of tumor volume, which was determined in each measurement using the following for-mula: volume (mm3) = (D2 x d) x 0.52, where (D) and (d) are the longest and shortest tumor diameters, respectively. After 27 days of nicotine treatment, all mice were sacrificed and their tumors excised, photographed, measured, fixed in 4% PFA and embedded in paraffin for subsequent immunohistochemistry (IHC) analysis.
    IHC assays to examine the expression of proliferation or angiogen-esis markers in the above tumor xenografts were performed on serial Sections (3.5 μm thick) using a Dako commercial kit (EnVision™ FLEX System kit) following the manufacturer’s instructions. Briefly, depar-affinized tissue slices were subjected to antigen retrieval and en-dogenous peroxidase blockade. Slices were then incubated with the anti-Ki-67 (Ready-to-Use) or anti-VEGF (dilution 1:50) primary anti-bodies followed by incubation with a secondary polyvalent antibody from Dako (goat anti-rabbit and anti-mouse Ig-HRP; 1:200). The target antigens marked by the respective primary antibodies showed the brown color generated by incubating slices with the EnVision™ FLEX DAB + Substrate Chromogen System (Dako). Sections were hematox-ylin and eosin counterstained using the EnVision FLEX Hematoxylin reagent and mounted with the Dako Mounting Medium. Section images were taken using a Nikon TE2000-S microscope with ImageJ software.
    2.10. Data and statistical analysis
    Data normalization was used for evaluation of the nicotine or NNK effects on cell migration measured by the transwell migration test (Fig. 3B) or on epithelial-mesenchymal transition measured by Western blot (Fig. 5B), where the untreated (blank or control) cell value was set to 100% or 1, respectively. The Mann-Whitney U test was used for comparisons between two groups, and one-way ANOVA followed by the Newman-Keuls post-hoc test (parametric data) for multiple comparisons (Fig. 6A and 6C). Data were represented as mean ± SEM of at least five independent experiments, except for Western blots in Fig. 1 (n = 2) and Fig. 5 (n = 3). A p value ≤ 0.05 was considered statistically significant. Statistical analyses were performed using Prism software, version 5 (GraphPad).
    3. Results
    3.1. Stable overexpression of dupa7-Myc in NSCLC cells does not alter the expression level of endogenous a7 mRNA or protein
    Cells from lung adenocarcinoma (A549) or squamous cell carcinoma of the lung (SK-MES-1) were used for these and the subsequent in vitro experiments since, in addition to representing the two major histolo-gical types of NSCLC, both cell types express α7-nAChRs whose acti-vation by nicotine or NNK promotes tumor growth and development [11,12,25,26]. The first experiments in this study were designed to rule out the possibility that dupα7 overexpression could inhibit the en-dogenous expression of α7 since, if this were the case, the interpretation of our results would change radically. Thus, we analyzed the expression of dupα7 and α7, at the mRNA or protein level, by qPCR and im-munocytochemistry combined with confocal microscopy or Western