br Generation of adducin overexpressing
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 . 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.
2.6. Cell fractionation and immunoblotting analysis
The nuclear and the cytoplasmic fractions of NSCLC cells were prepared using a NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher) according to manufacturer's instructions. To prepare total cell lysates, cells were homogenized using a Dounce homogenizer in RIPA buﬀer (20 mM Tris, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% sodium deoxycholate, 1% Triton X-100 (TX-100), and 0.1% SDS, pH 7.4) containing protease inhibitor cocktail and phosphatase in-hibitor cocktails 2 and 3 (Sigma-Aldrich). The obtained total cell lysates were cleared by centrifugation (20 min at 14,000 ×g), diluted with 2× SDS sample loading buﬀer and boiled. SDS-polyacrylamide gel elec-trophoresis was conducted using a standard protocol with equal amounts of total protein (10 or 20 μg) loaded per each lane. The se-parated proteins were transferred to nitrocellulose membranes, the membranes were blocked with either 5% non-fat milk for general protein detection, or with 3% bovine serum albumin (BSA) to detect phosphorylated proteins. The blocked membranes were incubated overnight with primary antibodies, exposed to HRP-conjugated sec-ondary beta-Nicotinamide for 1 h, and the labeled proteins were visualized using a standard enhanced chemiluminescence solution and X-ray films.
2.7. Scratch wound assay
Confluent cell monolayers were mechanically wounded by making a thin scratch wound with a 200 μl pipette tip. The bottom of the well was marked to define the position of the wound and the monolayers were supplied with fresh cell culture medium. The images of a cell-free area at the marked region were acquired at the indicated times after wounding using an inverted bright field microscope equipped with a camera. The percentage of wound closure was calculated using an Image J software (NIH, Bethesda, MD).
2.8. Boyden chamber migration assay
6. 5 mm membrane inserts with 8.0 μm pores (Corning Incorporated). The membrane inserts were coated with 15 μg/cm2 of collagen I. Cells were detached from the plate using a TrypLE Express solution (Thermo-Fisher), resuspended in serum-free medium, and added to the Transwell upper chamber at the density of 6000 cells per chamber. A complete cell culture medium containing 10% FBS as a chemoattractant was added to the lower chamber and cells were allowed to migrate for either
12 h, or 16 h at 37 °C. Membrane inserts were fixed with methanol and non-migrated cells were removed from the top of the filter using a cotton swab. The cells remained at the bottom of the filter or within the membrane were labeled with DAPI nuclear stain, visualized by a fluorescence microscope and counted by using the Image J software.
2.9. Matrigel invasion assay
The Matrigel invasion assay was performed using commercially available BD Biocoat invasion chambers (BD Biosciences). Cells were detached, resuspended in serum-free medium, and added to the upper part of the invasion chamber at the density of 10,000 cells per chamber. A complete cell culture medium containing 10% FBS as a chemoat-tractant was added to the lower chamber and cells were allowed to invade Matrigel for 24 h at 37 °C. The Matrigel plugs were washed with HBSS, fixed with methanol and non-migrated cells were removed from the top of the gel using cotton swabs. The invaded cells were stained with DAPI, visualized by a fluorescence microscope and counted by using the Image J program.