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  • α-CEHC BBA Molecular Cell Research br

    2020-03-24

     BBA - Molecular Cell Research 1866 (2019) 395–408
    2. Materials and methods
    2.1. Human gene expression analysis
    Gene expression profiles for human lung cancer samples were gen-erated by The Cancer Genome Atlas (TCGA). We utilized the RSEM-quantified RNA-seq data for lung adenocarcinoma (LUAD) patients made available by the Broad GDAC Firehose repository (http://gdac. broadinstitute.org). This dataset includes 576 samples (515 primary solid tumors; 59 normal lung tissue). In order to determine whether ADD1 or ADD3 are differentially expressed between tumor and normal tissues we removed 58 cases with paired controls to create independent groups and performed a Wilcoxon rank sum test on the +1-shifted log2 values.
    2.2. Antibodies and other reagents
    The following monoclonal (mAb) and polyclonal (pAb) α-CEHC were used to detect cytoskeletal, focal adhesion and other proteins: anti-ADD1 pAb and ADD3 mAb (Santa Cruz Biotechnology, Dallas, TX); anti-FAK, paxillin, E-cadherin and vimentin mAbs (BD Biosciences, San Jose, CA); anti p-FAK, FLAG, p-paxillin, Src, p-Src, cadherin-11 and GAPDH pAbs (Cell Signaling, Beverly, MA); anti-N-cadherin pAb (Abcam, Cambridge, MA); anti-P-cadherin mAb (Millipore, Billerica, MA). Alexa Fluor-488-conjugated donkey anti-rabbit, Alexa Fluor-555-conjugated donkey anti-mouse secondary antibodies, and Alexa Fluor-488 or Alexa Fluor-555-labeled phalloidin were obtained from Thermo-Fisher Scientific (Waltham, MA). Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies were obtained from Bio-Rad Laboratories. A functional inhibitory goat anti-human cadherin-11 antibody was obtained from R&D Systems (Minneapolis, MN) and control goat IgG was purchased from Jackson Immunoresearch Laboratories (West Grove, PA).
    Non-transformed and transformed HBEC3-KT and HBEC3-KTRL53Myc human bronchial epithelial cells were obtained from Dr. John D. Minna, The University of Texas Southwestern Medical Center. NCI-H1573, HCC4019, NCI-H1299, NCI-H2030, NCI-H1703 and NCI-H23 lung cancer cell lines were provided by Dr. Samir Hanash, The University of Texas MD Anderson Cancer Center, whereas 16HBE14o bronchial epithelial cells were provided by Dr. Dieter Gruenert, University of California San Francisco. NCI-H441 lung cancer cells were purchased from the American Type Culture Collection. 16HBE14o cells were cultured in MEM medium (Thermo-Fisher) supplemented with 10% fetal bovine serum (FBS), HEPES, penicillin and streptomycin. H441 cells were cultured in RPMI medium (Thermo-Fisher) supple-mented with 5% FBS, dexamethasone (250 μg/ml, Sigma-Aldrich), in-sulin-transferrin‑sodium selenite (Thermo-Fisher), penicillin and streptomycin. H1573, HCC4019, H1299, H2030, H1703 and H23 cells were cultured in RPMI medium supplemented with 10% FBS, HEPES, sodium pyruvate and antibiotics. HBEC3-KT and HBEC3-KTRL53Myc cells were cultured in Keratinocyte serum-free medium (Thermo-Fisher) without antibiotics. The cells were seeded on collagen-coated coverslips for immunolabeling experiments and on 6-well plastic plates for the functional and biochemical studies.
    2.4. CRISPR-Cas9 mediated knockout of ADD1 and ADD3
    A stable knockout of ADD1 or ADD3 in H1573 and 16HBE14o cells was carried out using a CRISPR-Cas9 technology. The guide oligonu-cleotide sequences used for knocking out ADD1 and ADD3 are shown in the Supplemental Table 1. The guide oligonucleotides were phos-phorylated, annealed and cloned into the BsmBI site of a lentiCRISPR v2 vector (Addgene, 52961) according to a published protocol [43,44].
    S. Lechuga et al.
    Obtained constructs were verified with sequencing. Transfer plasmids possessing annealed guide oligonucleotides were transformed into re-combination-deficient Stbl3 bacteria and amplified plasmids were iso-lated from the bacteria using Qiagen midi prep plasmid isolation kit. Lentiviruses were produced by transfecting HEK-293T cells with the transfer lentiCRISPR v2 plasmids and packaging plasmids pLTR-G (Addgene, 17532) and pCD/NL-BH DDD (Addgene, 17531). Viral su-pernatants were collected 48 and 72 h after transfection and used to infect H1573 and 16HBE14o cells. After 24 h of the infection, the len-tivirus-containing medium were replaced with fresh cell culture medium containing puromycin (2 μg/ml for H1573 cells and 10 μg/ml for16HBE14o cells) and puromycin-resistant cells were collected after 7 day selection.
    2.5. Generation of adducin-overexpressing lung cancer cells
    H1299 cell lines with stable overexpression of either ADD1, or ADD3 were generated using a lentiviral expression system. An expres-sion pLKO.AS2.neomycin vector encoding FLAG-tagged wild-type ADD1 was obtained from Dr. Hong-Chen Chen, National Chung Hsing University, Taiwan [45]. A cDNA encoding human ADD3 (clone BC062559 in the pOTB7 vector) was obtained from Transomic Tech-nologies (Huntsville, AL) and the ADD3 insert was cloned into the pLKO.AS2.neo vector. All obtained constructs were verified by se-quencing. H1299 cells grown till 70% confluency were infected with the lentiviral particle-containing supernatants. After 24 h infection, the medium was replaced with fresh cell culture medium containing 500 μg/ml neomycin and neomycin-resistant cells were selected for 7 days.