Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), calcium- and magnesium-free phosphate-buffered saline, L-glutamine and trypsin-versene mixtures were purchased from Biowhittaker (Walkersville, MD). Insulin (bovine) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St Louis, MO). Arecoline hydrobromide was purchased from Aldrich (Milwaukee, WI). The sources of other reagents are either listed below were of the highest purity available. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Invitrogen.MG132 and Dexamethasone were purchased from Sigma Chemical Co.
Ishikawa human endometrial adenocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA). Ishikawa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% Fetal bovine Serum, 10 μg/ml bovine insulin and 50 U/ml penicillin, 50 U/ml streptomycin and 2 mM Lglutamine. The cells were grown to subconfluency in a humidified air chamber at 37°C containing 5% CO2. Arecoline (99.9% high-performance liquid chromatography grade) was dissolved in appropriate concentrations in DMSO. DMSO was used as vehicle control for all experiments. All the experiments utilized cultured Ishikawa cells in passage 25 to passage 28.
Western Blot Analysis
After the indicated treatments, cells were harvested in radioimmune precipitation assay buffer (150 mM NaCl, 0.5% deoxycholate, 0.1% NoNidet-p40 (Nonidet P-40, Flulta Biochemitra, Switzerland), 0.1% SDS, 50 mM Tris) containing protease and phosphatase inhibitors (50 g/ml phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 5 g/ml leupeptin,0.1 g/ml NaF, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 0.1 mM_-glycerol phosphate). These extracts were then quantified using the Lowry Method (Bio-Rad Laboratories, Hercules, CA). Equal amounts of total cellular protein were mixed with loading buffer (25% glycerol, 0.075% SDS, 1.25 ml β-mercaptoethanol,10% bromphenol blue, 3.13% 0.5 M Tris-HCl, and 0.4% SDS (pH 6.8) and fractionated on 10% polyacrylamide/0.1% SDS resolving gels by electrophoresis. Spectra Multicolor Broad range Protein Ladder from Fermentas life sciences was used as the molecular weight standard. Proteins were electrically transferred to nitrocellulose membranes (Micron Separations, Inc., Westboro, MA). Equal protein loading was confirmed by Ponceau S staining of blotted membranes. Proteins were blocked for one and half hour at room temperature with Western wash buffer-5% NFDM (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20, 5% nonfat dry milk). Protein blots were subsequently incubated for overnight at 4 degree temperature with antibody in western buffer. The antibodies used were rabbit anti-ZO-1 (Invitrogen); rabbit anti-Claudin-1 (Santa Cruz Biotechnology); rabbit anti-E-cadherin (Santa Cruz Biotechnology); rabbit anti-beta-catenin (Santa Cruz Biotechnology); and rabbit anti-HER2/neu (Santa cruz Biotechnology). The working concentration for all antibodies was 1 μl/ml in Western wash buffer. Immunoreactive proteins were detected after incubation with horseradish peroxidase conjugated secondary antibody diluted to 0.25 μl/ml in Western wash buffer (goat anti-rabbit IgG and rabbit anti-mouse IgG (Bio-Rad). Blots were treated with ECL western blotting detection reagent (GE healthcare) and detected on the high performance chemiluminescence film (GE healthcare, UK).
Reverse Transcription PCR
Ishikawa cells were harvested in PBS and total RNA was isolated. RNA was quantified. 5 μg of total RNA was subjected to reverse transcription using murine myelogenous leukemia reverse transcriptase with First strand Buffer, random Primer (hexamers), dNTPs. 2 μl of cDNA was then subjected to PCR using Platinum Taq, 10 × PCR buffer, and 200 μM each dNTP (Invitrogen) along with the following primer sets and conditions: HER2 Forward 5'-CCAGCTCTTTGAGGACAACT - 3' and Reverse 5'-ATGTCCTTCCACAAAATCGT- 3', and the cycling conditions were 30 seconds at 95°C followed by 30 seconds at 52°C for annealing and finally 30 seconds at 72°C for extension for 26 cycles. ZO-1 Forward 5'-CGAGTTGCAATGGTTAACGGA-3' and Reverse 5' -TCAGGATCAGGACGACTTACTGG- 3', and the cycling conditions were 30 seconds at 95°C followed by 30 seconds at 55°C for annealing and finally 30 seconds at 72°C for extension for 26 cycles. GAPDH primers 5'-TGAAGGTCGGAGTCAACGGATTTG-3', GAPDH Reverse: 5'-CATGTGGGCCATGAGGTCCACCAC-3' (Ambion, Austin TX) served as a control, and PCR was performed according to the manufacturer's instructions. The PCR products were run on 1.1% agarose gels with Ethidium bromide along with a 1-kb plus DNA ladder (Invitrogen).
Indirect Immunofluorescence Assay
For indirect immunofluorescence assays, cells were grown on two well chamber slides from Nunc (Fisher scientific, Rochester, NY). The cells were fixed with 3.75% formaldehyde in PBS for 20 min on ice. After three additional washes with PBS, the plasma membrane was permeabilized with 0.1% Triton X-100; 10 mM Tris HCl at PH 7.5, 120 mM NaCl; 25 mM KCl; 2 mM EGTA; and 2 mM EDTA for 10 min at room temperature. Cells were incubated with 3% Bovine serum albumin (Sigma) in PBS before incubation with primary antibodies. Rabbit anti-ZO-1 antibody (61-7300 from Invitrogen) and rabbit anti-E-Cadherin (C212 from Santa Cruz Biotechnology) were used at a 1:400 dilution. Secondary Alexa 488 anti-rabbit (Molecular Probes, Inc., Eugene, OR) were used at a 1:400 dilution. Stained cells were mounted with Vectashield Mounting media containing DAPI (Vector Laboratories, Inc., Burlingame, CA). Stained and mounted cells were then processed with a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Thornwood, NY).
Transfection of Ishikawa cells
To generate stably transfected cells, Ishikawa cells at passage number 25, were transfected with either 0.2 μg of CMV-neo empty vector or CMV-HER2 (CMV empty vector and CMV-HER2 were generously provided by the laboratory of Dr. Bjeldanes, UC Berkeley, CA, USA), using polyfact (Qiagen, CA) and following the manufacturer's suggested protocol. Cells were fed 24 h after transfection with DMEM, supplemented with 10% fetal calf serum, penicillin/streptomycin. The media was replaced with same media containing 0.7 mg/ml G418 (neomycin analog, Mediatech, Herndon, VA) to select for transfected cells. Selection media was replaced every 24 hours for a month and surviving cell populations were propagated in selection media. Experimental treatments were not performed in selection media.
The nuclear and nonnuclear subcellular fractions were harvested from cell extracts using the NE-PER Nuclear Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) according to the manufacturer's instruction. The total protein was quantified using Bradford reagents (BioRad). Cell fractions were examined by Western blots as described above. Anti-lamin was used as a marker for nuclear fraction.