Cell lines and culture
HEK293T, BT20, MCF 7, MDA-MB-231 and MDA-MB-361 cells were maintained in Dulbecco’s modified Eagle’s medium, as described previously (17-19), AU565 cells were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 1% penicillin-streptomycin, and 10% fetal bovine serum (FBS, Gibco BRL) in a humidified atmosphere (37°C/5% CO2). HEK293T cells were passaged every second day, and other cells were passaged every third day, as described previously (17-19).
Determination of migration using wound healing assays
In the wound healing migration assays, AU565 or MCF 7 cells were seeded in 6-well plates at density a of 1x106 cells/well, incubated for 24 h and at 37 °C and scratched using a yellow pipette tip when the cells covered the well. as described previously (17-19). The cells were washed with PBS twice to clear the floating cells and treated with control siRNAs or LRIG1 siRNAs. Images were captured at 0, 24 and 72 h using a fluorescence microscope at 40x magnification. Subsequent recolonization of the stripped surface was quantitated by measuring the distance between wounded edges.
SDS-PAGE and immunoblotting
Equal quantities (measured in a Bradford Assay) of cell lysate (0.2% NP-40 (IGEPAL, Sigma-Aldrich)-TNE (10 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM EDTA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels. as described previously (17-19). Resolved proteins were then transferred to PVDF membranes and incubated with the appropriate primary antibodies (rabbit polyclonal anti-LRIG1 [1:1,000] (Abcam #ab36707), mouse monoclonal anti-Erbb2 [1:1,000] (Cell Signaling Technology #2242), rabbit polyclonal anti-AKT [1:1,000] (Cell Signaling Technology #9272S), rabbit anti-phospho-AKT (T308) [1:1,000] (Cell Signaling Technology #9275S), rabbit anti-phospho-AKT (S473) [1:1,000] (Cell Signaling Technology #9271S) mouse monoclonal anti-GSK-3β [1:1,000] (Santa Cruz #11B9), rabbit monoclonal anti-β-catenin [1:1,000] (Cell Signaling Technology #D10A8), or mouse anti-GAPDH (1:5,000; Santa Cruz; sc-32233), followed by anti-rabbit secondary antibodies coupled to horseradish peroxidase (1:5,000 dilution) (Thermo Fisher Scientific) or anti-mouse secondary antibodies coupled to horseradish peroxidase (1:5,000 dilution) (Thermo Fisher Scientific). The blots were then visualized by ECL. The relative band intensities were measured using ImageJ. 1.46r.
Erbb2 stability analysis
AU565 cells were treated with 50 μg/ml cycloheximide (Sigma; C1988-1G) 24 h after siRNAs treatment and then harvested at 0, 0.5, 1, or 3 h posttreatment.
Immunofluorescence analysis and confocal microscopy
AU565 (2×104) cells grown on coverslips in 24-well plates cells were fixed for 20 min with 4% paraformaldehyde, followed by washing three times in 1×PBS. Then, cells were permeabilized with 0.2% Triton×100 (0.1% saponin [Sigma-Aldrich #S7900-25G]/1% bovine serum albumin [Bovogen Biologicals #BSAS0.1]/0.1% sodium azide [Sigma-Aldrich #S2002-100G] in 1×PBS) for 20 min, followed by washing three times in 1×PBS, as described previously (17-19). Next, cells were incubated overnight at 4°C with anti-LRIG1 (1:300) and anti-Erbb2 (1:300) in permeabilization buffer. Immunofluorescence detection was performed using fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG + IgM (1:350; Jackson ImmunoResearch #115-095-044) and rhodamine (TRITC)-conjugated goat anti-rabbit IgG (1:350; Jackson ImmunoResearch #115-025-003) in permeabilization buffer at room temperature for 1 h. Cells were mounted with Fluoroshield mounting medium containing DAPI (Abcam #ab104139). Digital images of stained cells were captured under a confocal microscope (LSM710; Zeiss, Germany), as described previously (17-19).
To examine the physical interaction of LRIG1 and Erbb2 in MCF7 and AU565 cells, 1×106 cells on 10 cm plates were seeded, as described previously (17-19) For immunoprecipitation, rabbit polyclonal anti-LRIG1 antibody was added to the lysates, as described previously (17-19), followed by immunoblotting with mouse monoclonal anti-Erbb2.
Patients and tissues
Fibroadenoma and Invasive ductal tissues were obtained from 70 different patients (male and female) from Armed Force Institute of Pathology, (AFIP) Rawalpindi, Pakistan. Information on patient demographics (gender, age), tumor features (size of tumor, histological grade, lymph node metastasis, Nottingham index), hormones receptor status (Estrogen and Progesteron) and Her2/ neu status were obtained from hospital records. Study was approved by AFIP and Atta-ur-Rahman School of Applied Biosciences Ethics and Investigation Committee. Biopsies were taken after patient’s informed consent. These biopsies were formalin fixed; paraffin embedded for immunohistochemistry.
In order to determine the expression level of LRIG1 in paraffin embedded and formalin fixed tissue biopsies, immunohistochemistry was done by the protocol mentioned on catalogue of anti-LRIG1 antibody ab36707. The 5µm thick tissue sections were incubated with rabbit polyclonal anti-human/mouse LRIG1 antibody (ab36707; Abcam, UK) at 4°C overnight without performing antigen retrieval step. The HRP labeled goat anti rabbit secondary antibody (ab6721; Abcam, UK) was applied and incubated for 1 hour at room temperature. DAB staining kit (Abcam Cat: ab64238, UK) was used for detecting the expression of LRIG1. Tissue sections were counterstained with hematoxylin, dehydrated and mounted via mounting media. Negative control was PBS without the primary antibody and positive control was normal breast tissue.
Evaluation of Immunohistochemical Staining
Each slide was evaluated by two histo-pathologists who were unaware of clinical outcome and results of the study. Positive staining was defined as brown color staining on the glandular epithelium of normal breast tissue and was measured by counting the positive cells at 400X in three representative high-power fields for each section by using Labomed TCM400 inverted microscope (Labi America Inc., USA) and stained sections were photographed using ProgRes Capture Pro 2.6 (JENOPTIK Laser, Optik, Systeme GmbH, Germany). Immunoreactivity was scored as a). Zero or (-ve) for sections that have less than 10% positive cells. b). One or (+ve) for sections having more than 10% positive cells. Intensity was scored as a). Zero or (-ve) for no staining, b). One or (+ve) for low staining, c). Two or (++) for moderate staining, c). Three or (+++) for high or normal staining (20).
The history of patient such as age, Nottingham grading index, histological findings and Her2/neu status were included in study. Spearman’s Rank Order Correlation was used to determine the relationship between expression of LRIG1 and history of patients. One Sample T-test was run in order to evaluate the correlation between the risk factor fibroadenoma and disease IDC. Data are expressed as the mean ± standard deviation. Mean values were compared using Student’s t-test. P- values less than 0.05 were taken as statistically significant in correlation. Statistical analysis was performed using Statistical Package for the Social Sciences, version 16.0 (SPSS Inc, Chicago, IL, USA).
The experiments performed in current study were in accordance with the relevant guidelines and regulation and approved by institutional review board and ethical review committee of National University of Sciences and Technology.