br Real time PCR was carried out on Light Cycler
Real-time PCR was carried out on Light Cycler 480 II machine (Roche) using SYBR Green Chemistry. The oligonucleotide primer se-quences used for real-time PCR analysis are listed in Table 1. For all the above genes the amplification conditions were as follows: initial de-naturation; 95 °C for 10 min and cycling steps of denaturation 95 °C for 20 s, annealing at 600C for 20 s and extension at 720C for 20 s, repeated for 40 cycles. This was followed by melting reaction step at 950C and 650C for 10s and 60s respectively and data acquisition at 950C for 1 s. β –actin was used as a reference gene for normalization and relative change in gene expression was analyzed using 2-ΔΔCT method.
2.4. Matrigel invasion assay
In vitro transwell cell invasion was performed using Matrigel in-vasion chamber (BD Biosciences). Breast cancer 94079-80-8 MCF-7 and MDA-
Ets-1 5′-GCAGCCAGTCATCTTTCAACAGCC5′-3′ 5′-TCAGCACGGTCCCGCACATA-3′ MMP-9 5′-CTGGAGACCTGAGAACCAA-3′ 5′-ACTGCTCAAAGCCTCCACAAGA-3′ N-cadherin 5′-CCTCCAGAGTTTACTGCCATGAC-3′ 5′-GTAGGATCTCCGCCACTGATTC-3′ Vimentin 5′-AGGCAAAGCAGGAGTCCACTGA-3′ 5′-ATCTGGCGTTCCAGGGACTCAT-3′ Slug 5′-ATCTGCGGCAAGGCGTTTTCCA-3′ 5′-GAGCCCTCAGATTTGACCTGTC-3′ Snail 5′-TGCCCTCAAGATGCACATCCGA-3′ 5′-GGGACAGGAGAAGGGCTTCTC-3′ β –actin 5′-GGATGCAGAAGGAGATCACTG-3′ 5′-CGATCCACACGGAGTACTTG-3′
MB-231(3× 104) were plated in 6-well plates and transfected with Ets-1 siRNA as described above for 24 h. After 24 h homogenous cell sus-pension was added (30,000 cells/insert) in each insert containing 8.0-μm polycarbonate membrane and allowed to invade for 24 h. Media was aspirated and non-invasive cells were removed gently with a cotton swab dipped in alcohol. The cells attached at the bottom were stained with 5% crystal violet and visualized under a light microscope (Olympus, 1X81, Japan). Each insert was later transferred to empty wells containing 200 μl RIPA buffer, incubated for 1 h and 100 μl from each well was taken in a 96-well plate. Quantification was done by taking OD at 560 nm in a plate reader (MULTISKAN GO, Thermo Scientific USA).
2.5. Immunofluorescence microscopy
Immunofluorescence microscopy was performed on MCF-7 and MDA-MB-231 breast cancer cells. Cells (1× 104) were cultured on coverslips in a 6-well plate and treated for 48 h as described in Ets-1 siRNA transfection experiment. The medium was removed and cells were washed in 1× PBS (Phosphate Buffer Saline). The cells were then fixed in 100% methanol (chilled at −20 °C) at room temperature for 5 min. This was followed by washing three times with 1× PBS and permeabilization with PBS containing 0.2% Triton X-100 for 10 min. The cells were again washed in 1× PBS three times for 5 min and in-cubated with 1% BSA, in PBST (PBS+ 0.1% Tween 20) for 30 min to block non-specific binding of the antibodies. Cells were then incubated in 1% BSA PBST solutions of polyclonal rabbit antibodies against Ets-1 (ab26096, Abcam) and MMP-9 (ab38898, Abcam) for overnight in a humidified chamber at 4 °C. Next day cells were washed three times in PBS for 5 min each and incubated with FITC labelled goat anti-rabbit secondary antibody (ab6717, Abcam) against Ets-1 and MMP-9 for 2 h at room temperature in dark. Cells were then washed with PBS three times for 5 min and stained with 1 μg/ml DAPI/well. The cells were rinsed with PBS and coverslips mounted on slides with a drop of mounting media. The cells were then visualized under fluorescent mi-croscope (Olympus, 1X81, Japan).
2.6. Western blotting
Breast cancer cell lines MCF-7 and MDA-MB-231 were transfected with Ets-1 siRNA for 48 h. Cells were then harvested, rinsed with 1× PBS and lysed using RIPA lysis buffer (1 M Tris-HCl, 5 M NaCl, 0.1 M PMSF, 0.5 M EDTA, 10% Triton-X-100, 10% Sod. Deoxycholate, 10% SDS, 0.1 mg Protease Inhibitor and ddH20) and centrifuged at 14,000 rpm at 4 °C for 20 min. Supernatant containing the whole cell lysate (WCL) was collected and total protein concentration was mea-sured using standard BCA assay (Thermo Scientific, USA) and Qubit 3.0 Fluorometer (Thermo Fisher, USA). 50 μg of WCL was loaded per lane and electrophoresed on 10% SDS-PAGE gels and then electrotransferred in semi-dry transblotter (Bio-rad) to Immobilon-PSQ membranes (Millipore Corporation, Bedford, MA). 1:1000 dilution of polyclonal rabbit primary antibodies against Ets-1 (ab26096, Abcam) and MMP-9 (ab38898, Abcam) were then used and incubation was carried out overnight in 1× PBS supplemented with 5% skimmed milk powder, 0.05% Tween 20 (Sigma-Aldrich, USA). Visualization of the bands was done using goat anti-rabbit immunoglobulin G (IgG) secondary anti-body conjugated with horseradish peroxidase (Santa Cruz Biotech) at a dilution of 1:5000 by chemilumniscent Amersham ECL™ detection kit for western blotting (GE Healthcare) using blots (as X-ray) which were exposed for different time intervals. After exposure, blots were devel-oped in developer and fixed (Carestream, USA). Stripping of the membrane and re-probing with β-actin was done to confirm equal loading and normalization.