br Cell transfection and selection of stable dupa
2.2. Cell transfection and selection of stable dupa7-Myc transfectants
The dupα7.pcDNA3.1/Myc-His construct used for transfection was prepared in our laboratory as described elsewhere ; it contains the full-length human dupα7 cDNA sequence in frame with the Myc-His tag. The A549 or SK-MES-1 KN93 were transfected either with the above plasmid or with the corresponding empty-vector (pcDNA3.1/Myc-His) using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer´s instructions. Forty-eight hours after transfection, the cells were trypsinized and plated in culture medium supplemented with the aminoglycoside antibiotic, geneticin (600 μg/ml; Sigma-Aldrich), for the selection of stably-transfected cells (A549dupα7 or SK-MES-1dupα7). Positive clones were double-confirmed by: 1) quantitative real-time PCR (qPCR) as detailed below; or 2) immunocytochemistry com-bined with confocal microscopy using the anti-Myc (1:200) primary antibody followed by the Alexa Fluor 488 goat anti-mouse IgG [1:400; Molecular Probes (Invitrogen, USA)] secondary antibody as described elsewhere .
2.3. qPCR assay of nAChR subunit gene expression
Techniques for RNA extraction and α7 or dupα7 gene expression analysis from cells or tumor tissues by qPCR from reverse-transcribed RNA using the SYBR green-based assays (Bio-Rad, Hercules, CA) and the ABI Prism 7500 Sequence Detector (Applied Biosystems, Foster City, CA), have been described elsewhere [9,20,21]. The following set of primers were used for PCR amplification of the corresponding tran-script: α7, forward 5´-GCTGCAAATGTCTTGGACAGAT and reverse 5´-AACAGTCTTCA−CCCCTGGATAT; dupα7, forward 5´- CAATTGCTAA TCCAGCATTTGTGG and reverse 5´−CCCAGAAGAATTCACCAACACG. The next pair of primers [forward 5′-TGATCAAGGGAAAGATGACCA and reverse 5′-AACCCTCTTGCAATCGAAAA] was employed to amplify the human D esterase gene (ESD), which was selected as an endogenous control for the PCR reaction since, among the housekeeping genes, that gene seems to have the most stable expression in NSCLC [22,23]. Cy-cling conditions for PCR were: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. Analysis of the melting curves de-monstrated that each pair of primers amplified a single product. Re-
lative changes in α7 or dupα7 mRNA expression in A549dupα7 or SK-MES-1dupα7 cells were assessed by the 2− Ct method using the corre- sponding mRNA expression in non-transfected cells (A549 or SK-MES-1) as calibrator (set to a value of 1).
2.4. Wound-healing assay in vitro
This assay serves to detect cell migration. Control or transfected NSCLC cells were seeded in 6-well plates (3.5 × 105 or 8 × 105 cells per well, respectively) and grown to 90% confluence (≈ 24 h). Subsequently, the cell monolayer was scratched vertically with a sterile
pipette tip and the wells washed with PBS to remove the detached cells. The cells were then incubated or not with nicotine, NNK or 10% FBS (positive control) to induce cell migration. Images of the wound were captured at and 24 h after scratching using a DXM1200 F digital camera attached to a personal computer, with a Nikon TE2000-S mi-croscope.
2.5. Transwell assay in vitro
The cell migration assay was also performed using the two-well Transwell Boyden Chamber (Corning-Costar, Cambridge, MA, USA) with a polycarbonate membrane of 8 μm pores per the manufacturer’s instructions. A total of 5 × 104 cells were placed in the upper chambers in serum-free medium containing or not nicotine or NNK; the lower chambers were loaded with complete medium (10% FBS). After 24 h, the non-invasive cells retained in the upper chambers were removed with a cotton swab, while the cells that had migrated across the base-ment membrane and attached to its lower face were fixed and their nuclei stained with DAPI before cell counting with the ImageJ software of the confocal microscope Leica TCS SP5.
2.6. Cell proliferation assay in vitro
Cell proliferation was evaluated using the Click-iT EdU Alexa Fluor 647 Imaging Kit (Life Technologies/Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, control or trans-fected A549 or SK-MES-1 cells were plated on 12-mm glass coverslips (1.1 × 104 or 1.7 × 104 cells/coverslip, respectively) and maintained in a serum-starved medium for 24 h. Then cells were incubated with 5-ethylnyl-2´-deoxyuridene (EdU) for 24 h while undergoing treatment with nicotine, NNK or 10% FBS (positive control) to induce prolifera-tion. Subsequently, cells were fixed, permeabilized and incubated with the Click-iT reaction cocktail to detect the EdU signal before staining nuclei with DAPI. The coverslips were visualized in a Leica TCS SP5 confocal microscope that automatically counted the number of EdU-positive cells in the microscope field with the ImageJ software.