The cell lines used in this study involved the negative control MCF-10A (non-tumorigenic epithelial breast cell line) and the breast cancer cell lines MCF-7, T-47D, MDA-MB-468, and MDA-MB-231. The aforementioned cell lines were purchased from Sigma-Aldrich, UK. In addition, Jurkat cells (leukemia T-lymphocytes) were used as a positive control and presented as a gift from Professor Matthew Holley, Department of Biomedical Science, The University of Sheffield, Sheffield, UK. The MCF-10A cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) containing 4.5 g/L glucose with l-glutamine, and supplemented with 1× non-essential amino acids (NEAAs; Bio Whittaker), 10 μg/mL epidermal growth factor (Sigma-Aldrich), 50 μM hydrocortisone (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), 0.1 μg/mL cholera toxin (Calbiochem), and 5% horse serum (Invitrogen).
The breast cancer cell lines were grown in DMEM (Lonza) containing l-glutamine with 4.5 g/L glucose and supplemented with 10% fetal calf serum (FCS; Seralab) and 1× NEAAs (Bio Whittaker). Regarding the positive control, Jurkat cells, they were grown in RPMI 1640 (Roswell Park Memorial Institute medium; Lonza) containing l-glutamine, and provided with 1× NEAAs and 10% FCS. Before use, the above media, Trypsin-Versene (EDTA) and phosphate-buffered saline (PBS) were warmed in a 37 °C water bath for at least half an hour.
Protein lysates preparation and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Whole protein lysates were extracted from the cell pellets of MCF-10A, Jurkat, MCF-7, T-47D, MDA-MB-231, and MDA-MB-468, using 1× lysis solution (5× RIPA buffer diluted with ddH2O), which was supplemented with phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich; 1 mM), nuclease (25 U/μL; Novagen), 1× each of two phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich), and protease inhibitors (Calbiochem). Cell pellets were re-suspended in 1-pellet volume lysis solution, pipetted up and down, incubated on ice for half an hour, vortexed each 10 min, and spun down for 10 min at 13,400 rpm using Mini Spin placed in 4 °C. Finally, the concentration of the supernatant, which represented the whole cell protein lysate, was determined, aliquoted, and kept in − 80 °C for long-term storage.
The protein concentration of cell lysates was determined by using Bio-Rad Protein Assay as per the manufacturer’s instructions. The assay involved preparing different dilutions of bovine serum albumin (BSA; stock 0.1 mg/ mL) to make protein standard curve. A set of Eppendorf tubes was prepared to contain five different amounts of ddH2O (800, 790, 750, 700, 650, and 600 μL) and mixed with standards’ amounts of freshly made BSA (0, 10, 50, 100, 150, and 200 μL, respectively). The BSA protein concentration in these tubes ranged from 0, 1, 5, 10, and 15 to 20 μL, respectively. Simultaneously, another set of Eppendorf tubes containing 800 μL of ddH2O each was used and mixed with 1 μL of each protein sample. Then, 200 μL of Bio-Rad dye reagent concentrate was mixed with all BSA standards and protein samples. After 5-min incubation at room temperature, 200 μL of each standard and sample was moved to 96-well plate to be read using plate reader. The optical density (OD) of all proteins was measured at 595 nm, and a standard curve was made using Microsoft Excel by plotting the OD of the standards against their concentrations. Finally, the protein concentration of each sample was calculated based on the equation of the standard curve.
Subsequently, the same concentration (30 μg) of each protein lysate was mixed with 5× sample loading buffer and diluted with ddH2O up to 22 μL, and each was boiled at 95 °C for 5 min by using heat block (Grant Instruments). Then, the protein samples along with pre-stained protein ladder (Geneflow) were spun briefly and loaded onto Sodium dodecyl sulfate polyacrylamide gel to run an electrophoresis called SDS-PAGE, which was performed to separate the protein samples. The gel was made up of 8% resolving gel and 5% stacking gel. Then, the electrophoresis was run at 130 V for 1 h at room temperature using 1× running buffer.
Western blotting (WB)
Immunoblotting (WB) was carried out to analyze the protein level of STAG3 in the cancer cells. Following SDS-PAGE, the samples were transferred from the gel to a nitrocellulose membrane (Amersham) assembled with blotting papers inside Mini Trans-Blot® Electrophoresis Transfer cell system (Bio-Rad) filled with Towbin transfer buffer. Ponceau S stain (Sigma-Aldrich) was used to check for the transfer efficiency from the gel to the membrane. In this step, the membrane was cut into suitable parts to enable each part to be probed later with the proper antibody. Later, the membrane was rinsed with tap water to get rid of the stain. Following blocking with 5% milk/PBST at room temperature for 1 h, the appropriate part of the cut blot was probed with either Abcam (Cat. No. ab69928), Sigma (Cat. No. HPA049106) anti-STAG3 antibody, or the control anti-β-actin (Abcam ab8226) or β-tubulin (Sigma-Aldrich T8328) antibodies, which were used as a loading control to confirm that the same amount of protein was loaded into each well of the gel. These primary antibodies were diluted in 5% milk/PBST and incubated with the blots overnight on a shaker at 4 °C. After washing three times with 0.1% PBST for 8 min each, a suitable secondary IgG horseradish peroxidase-linked antibody diluted in 5% milk/PBST was used. The secondary antibody was incubated with the membrane on a shaker at room temperature for 1 h. Washing was done as mentioned before, and the blots were incubated with 2 mL ECL detection reagents (GE Healthcare) for 1 min. Eventually, the membrane was exposed to X-ray films (Fuji Medical X-ray film), developed and fixed by Konica SRX 101A Processor to visualize the protein bands.
To explore which band was STAG3 protein on WB, transfection experiments were performed to knockdown STAG3. Small interfering RNA (siRNA) specific for STAG3 mRNA (si-STAG3) was used to transfect the breast cancer cell line MCF-7 cells. In this study, specific si-STAG3#1 sense GCGCAAGACCCAAGCCGAU and si-STAG3#2 sense UGACUAUGGUGACAUUAUC (Eurofins) were used. As a control, small-interfering ribonucleic acid (siRNA) non-specific for any gene termed scrambled (sense UAAUGUAUUGGAACGCAUA) from Eurofins was used to transfect the cells. Transfection conditions were optimized for the above cell line. Standard transfection in six-well tissue culture plates was performed. The optimized cell numbers (2 × 105 in 2 mL media) were plated overnight using complete medium without antibiotic. The cells were approximately 50–70% confluent at the time of transfection. The next day, if necessary, the media was substituted with fresh complete media. Otherwise, the transfection continued by the addition of the optimized amount of siRNA to DMEM serum-free medium (SFM) in an Eppendorf tube, which was left for 5 min at room temperature. Then, appropriate amount of Dharmafect® #4 (transfection reagent; Thermo Scientific) was added to SFM in an Eppendorf tube and left for the same time. After that, siRNA-SFM was mixed by pipetting with the Dharmafect-SFM and kept for approximately 25 min at room temperature. A suitable amount of the mixture (siRNA-DharmaFECT-SFM) was added dropwise to each well containing the growing cells. Finally, the plates were incubated at 37 °C, 5% CO2 in a humid incubator for 24 or 48 h of transfection before collecting cell pellets to be used in western blot.
Protein lysates preparation and immunoprecipitation (IP)
For the IP assay, the cells were grown to 85% confluence in 10 cm cell culture dishes. One dish was used for the control and other two dishes for each primary antibody to be used. Later on, the media was removed from all plates, and the cells were rinsed gently twice in ice-cold PBS. After discarding all of the PBS, 800 μL of lysis buffer (1% Triton-X100, 50 mM Tris pH 7.6, 200 mM NaCl, 1 mM EDTA, protease inhibitor, phosphatase inhibitor, benzonase) was added to each dish. The cells were scraped into lysis buffer, collected, and transferred to a 1.5-mL Eppendorf tube, which was incubated on ice for 20 min. At 13,000 rpm, cell lysates were spun down for 20 min. Next, protein G beads were washed in a 1.5-mL Eppendorf tube in 1 mL lysis buffer three times (spinning down at 3000 rpm for 30 s each time). One more spinning was done to remove the remaining liquid. Subsequently, an equal volume of lysis buffer was mixed with the beads. To set up the IP, aliquots of 40 μL of bead solution were dispersed into individual tubes, which were kept on ice. When the lysates were spun down, 50 μL of each lysate was used as an input sample, this would show whether the protein is present in the lysate and allow lining up any immunoprecipitated band. In order to perform WB analysis, an appropriate loading buffer was added in a suitable concentration to the lysates. The mixture was boiled at 95°C for 5 min and put in freezer at − 80°C till use. Then, the rest of the cell lysates were put in the appropriately labeled tubes containing protein G beads. While the tubes were kept on ice, 2–5 μg of IgG protein was added to the control sample and an equal mass of STAG3 antibody for the IP. Finally, all the samples were incubated on the cold room rotator at 4 °C and 20 rpm overnight. The next day, the samples were washed by spinning at 3000 rpm for 30 s, removing supernatant without touching the beads and replacing with 1 mL lysis buffer. This step was repeated four times, and the last time included spinning down and removal of liquid as much as possible without disturbing beads. At the end, 1× WB loading buffer (50 μL) was added and boiled for 5 min at 95 °C, which was followed by spinning down at 3000 rpm for 30 s. The supernatant was spun again, and 30 μL from which were loaded onto SDS-PAGE. With every sample to be loaded on the gel, inputs as well as the IgG control and STAG3 IP samples were also loaded. Both SDS-PAGE and WB were performed as described above.
Bioinformatics analysis of STAG3 isoforms
Using the online NCBI (National Center for Biotechnology Information) and Ensembl software, the Homo sapiens STAG3 isoforms were searched. The three-dimensional (3-D) structure of the isoforms was detected using Phyre2 software. Using PSORTII and SOPMA tools, the subcellular localization and the secondary structures of the isoforms were studied, respectively. Some of the physicochemical properties of the STAG3 proteins were determined by ProtParam software.