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  • br Immunofluorescence labeling and confocal microscopy br Cells


    2.10. Immunofluorescence labeling and confocal microscopy
    Cells cultured on collagen-coated coverslips were fixed with 4% PFA for 20 min at room temperature and permeabilized with 0.5% Triton X-100 for 5 min. Fixed and permeabilized cells were blocked with Hank's Balanced Salt Saline (HBSS) containing 1% BSA for 1 h at room tem-perature, followed by sequential 1 h incubations with primary anti-bodies and Alexa Fluor-conjugated secondary Pilocarpine diluted in the blocking solution. The immunolabeled coverslips were mounted using the ProLong® Gold Antifade reagent (Thermo-Fisher). Fluorescently labeled cell monolayers were examined using either a Zeiss LSM700 laser-scanning confocal microscope (Zeiss Microimaging, Thornwood, NY), or Leica SP8 confocal microscope (Wentzler, Germany). The images were processed using Zen Lite software (Carl Zeiss Microscopy LLC) and Adobe Photoshop.
    2.11. Extracellular matrix adhesion assay
    Cells were detached from the plate, counted with a hemocytometer, and resuspended in the complete medium. 10,000 cells were seeded to each well of a 24 well plate coated with either collagen I, or fibronectin and were allowed to adhere for 30 min at 37 °C. After incubation, un-attached cells were aspirated and the wells were gently washed with HBSS buffer. The attached cells were fixed with methanol and stained using a DIFF stain kit (IMEB Inc., San Marcos, CA). Images of adherent cells were captured using a bright-field microscope and the number of adhered cells was determined using Image J software.
    2.12. Cell proliferation assay
    Cell proliferation was examined by the MTT assay involving the conversion of the water-soluble MTT (3‑(4,5‑dimethylthiazo-l‑2‑yl)‑2,5‑diphenyltetrazolium bromide) into insoluble formazan. Cells were cultured on 98 well plates, and at different times post-plating, their medium was replaced with 100 μl of fresh culture medium con-taining 50 μg/ml of MTT. MTT added to 100 μl of medium alone was included as a negative control. Cells were incubated at 37 °C for 4 h and the generated formazan was dissolved by adding 50 μl of DMSO with 10 min incubation at 37 °C. The absorbance of formazan solution was
    S. Lechuga et al.
    measured using a standard plate reader at 540 nm.
    siRNA-mediated knockdown of Src was performed by using a spe-cific Dharmacon siRNA Smartpool (Thermo-Fisher) with non-targeting siRNA number 2 served as a control. Cadherin-11 was depleted by using individual siRNA duplexes and a negative siRNA control obtained from Qiagen. Cells were transfected using DharmaFECT 1 transfection re-agent in Opti-MEM I medium (Thermo-Fisher) with final siRNA con-centration for each target at 50 nM. Cells were used in experiments on days 3 and 4 posttransfection.
    2.14. Statistical analysis
    All data are expressed as means ± standard error (SE) from three biological replicates. Statistical analysis was performed by using a one-way ANOVA to compare the control and two experimental groups (knockout with two different adducin sgRNAs). A post-hoc t-test was used to compare controls with each adducin-depleted group. A two-tailed Student t-test was used to compare the results obtained with two experimental groups (control and adducin-overexpressing cells). We accounted for multiple comparisons by adjusting the significance level using a Bonferroni Correction. p values < 0.05 were considered sta-tistically significant.
    3. Results
    3.1. Adducins are either downregulated or mislocalized in mesenchymal-type lung cancer cells
    Despite early in vitro and animal model studies suggesting associa-tion of tumor progression with altered levels and localization of ADD1 and ADD3 [28,46], little is known about expression of adducins in human cancers. Therefore, we used the TCGA database to examine expression of ADD1 and ADD3 in clinical samples of patients with NSCLC. Fig. 1A demonstrates that mRNA levels were significantly lower in lung adenocarcinoma compared to normal lung tissue for both ADD1 (p < 10−15) and ADD3 (p < 10−5). Such expressional down-regulation was especially vivid for ADD1. NSCLC is characterized by significant phenotypic heterogeneity, which can be grouped into three broad categories: epithelial, mesenchymal and epithelial-mesenchymal hybrid [47]. The former category includes cells that preserve char-acteristics of well-differentiated normal pulmonary epithelium such as high E-cadherin expression, assembly of robust intercellular junctions and poor invasiveness. By contrast, mesenchymal-type NSCLC cells lost cell-cell contacts and acquired high invasiveness [47]. Therefore, we next sought to compare expression and localization of adducins in a panel of NSCLC cells with either the epithelial (H441, H1573, HCC4019) or the mesenchymal (H1299, H2030, H1703, H23) pheno-types. The epithelial panel also included non-transformed 16HBE14o human bronchial epithelial cells [48]. The epithelial and the me-senchymal phenotypes of these cells was confirmed by observing ex-pression of either E-cadherin or vimentin, respectively (Fig. 1B). Im-munoblotting analysis demonstrated that expression of ADD1 protein did not depend on cell phenotype, whereas the level of ADD3 protein was dramatically decreased in mesenchymal-type NSCLC cells (Fig. 1B). We also compared expression of adducins in the cell pair consisting of immortalized, non-transformed human bronchial epithelial cells, HBEC3-KT, and HBEC3-KTRL53M cells fully transformed by p53 knockdown and overexpression of KRAS G12V and c-Myc [49]. Transformation of HBEC cells caused a robust epithelial-to-mesench-ymal transition that was accompanied by a selective downregulation of ADD3 expression (Suppl. Fig. 1A).