Cell culture and Reagents
MCF7, T47D, and BT474 cells were procured from ECACC (Salisbury, Sussex, UK). MCF7 cells expressing Y537S and D538G mutants were kind gifts from Dr. Steffi Oesterreich (University of Pittsburgh, USA). HEK 293T cells were obtained from the Indiana University National Gene Vector Biorepository (Indianapolis, IN, USA). Cells were maintained in DMEM supplemented with 10% foetal calf serum (FCS), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C with 5% CO2. MCF7 cells expressing Y537S and D538G mutants were cultured using DMEM and 5% FCS. FCS and charcoal-stripped serum (CSS) were purchased from Labtech International (Sussex, UK). Brefeldin A (BFA), Thapsigargin (TG), Tunicamycin (TM), STF083010, and Blasticidin were purchased from Tocris (Bio-Techne Ltd, Abingdon, UK). Tamoxifen, fulvestrant, and AZD9496 were purchased from MedChem Tronica, Sollentuna, Sweden. MLN4924 was from EMD Millipore, Wicklow, Ireland. All other chemicals were obtained from Sigma-Aldrich, Wicklow, Ireland, unless otherwise stated.
CRISPR guide RNA encoding plasmids (Cat# KN201959) targeting XBP1 were purchased from Origene (Cambridge Biosciences, Cambridge, UK). The following plasmids: PCLXSN (Cat# 12343), 5myc-CDC6-WT (Cat# 109332), 5myc-CDC6-mut (Cat# 109333), psPAX2 (Cat# 12260), PMD2.G (Cat# 12259) and pUMVC (Cat# 8449) were obtained from Addgene, Watertown, MA, USA. Null control and V5-tagged RRM2 expressing lentiviral plasmids were obtained from the DNASU Plasmid Repository (Arizona State University, USA).
Generation Of Xbp1 Knockout Clones
Parental MCF7 cells were electroporated with two gRNA plasmids (5’-ACTTTAGGGGTCCCGTCGGC-3’ and 5’-CCCGTCGGCCGGGTTCGGCG-3’) targeting XBP1 and Cas9 plasmid using ‘Nucleofector Kit V’ (Cat# VCA-1003) and 4D nucleofector (Lonza) following the manufacturer's instructions. Transfected cells were selected using puromycin (1 µg/ml), single cell clones were isolated, and loss of XBP1 was assessed by western blotting and PCR.
Generation of RRM2 and CDC6 overexpressing clones
RRM2 overexpressing clones were generated using lentiviral transduction. Positive clones were selected using blasticidin containing media (5 µg/ml) for one week. CDC6 overexpressing clones were generated using retrovirus transduction followed by G418 (800 µg/ml) selection for two weeks.
Rna Extraction, Cdna Synthesis, And Qpcr
The qRT-PCR used here has been described previously . Briefly, Total RNA was isolated using Trizol (Invitrogen) and cDNA synthesis was carried out using ImProm-II™ Reverse Transcription System (Promega). Expression of genes of interest was carried out using predesigned prime time qPCR assays (Integrated DNA Technologies, Belgium) and StepOnePlus thermocycler (Applied Biosystems). Relative expression was calculated using the 2−ΔΔCT method.
Cell Proliferation Assay
MTS cell viability assay has been previously described . Briefly, cells (2,000 cells/well) were plated in 96-well plate using DMEM media containing 10% FCS. After indicated days, MTS + PMS was added into each well and incubated at 37oC for 4h. The O.D was measured at 490 nm.
Rabbit β-Actin antibody (Cat# A5060) was from Sigma-Aldrich (Wicklow, Ireland). XBP1s antibody (Cat# 647502) was obtained from Biolegend, London, UK. ER-α antibody (Cat# sc-8002), RRM2 antibody (Cat# sc-376973), CDC6 antibody (Cat# sc-9964) were purchased from Santa Cruz Biotechnology, Inc. (Bergheimer, Heidelberg, Germany. HRP linked anti-rabbit (Cat# 7074S) and anti-mouse (Cat# 7076S) secondary antibodies were purchased from cell signalling technology (Frankfurt, Germany).
The western blotting procedure used here has been described previously . Briefly, protein samples were run on SDS-PAGE and transferred into the nitrocellulose membrane. Blocking was done using either 5% non-fat dry milk in phosphate-buffered saline (PBS)/0.05% tween-20 (for ERα, XBP1s, β-Actin, and CDC6) or 2.5% milk + 2.5% bovine serum albumin in Tris-buffered saline (TBS)/0.1% tween-20 (for RRM2) for 2h at room temperature. After blocking, the membranes were incubated with primary antibody, ERα (1:1,000), XBP1s (1:1,000), CDC6 (1:500), Beta-Actin (1:2,000), and RRM2 (1:250) at 4oC overnight. After washing the membranes were incubated with appropriate HRP-conjugated anti-rabbit (1:10,000) or anti-mouse (1:5,000) antibody. The membranes were developed using Western Lightning® Plus-ECL, Enhanced Chemiluminescence Substrate (PerkinElmer, Llantrisant, UK) to visualise the protein bands.
Cell Death Analysis
This has been described previously . Cells (0.2 x 106) were plated in a 6-well plate. After 24h, cells were treated with either vehicle (0.1% DMSO) or with the required compounds for the indicated time points. The media was collected into a separate 15 ml tube, and cells were collected by trypsinization. The cells were then pelleted down by centrifugation at 1,200 rpm for 5 min at 4oC, and media was discarded. Cells were then washed once with ice-cold PBS and resuspended in fluorescent activated cell sorting (FACS) buffer (100–150 µl) and transferred into a 1.5 ml Eppendorf tube. Before analysis, 4 µl (stock conc. 0.5 mg/ml) of propidium iodide (PI) was added to 100 µl of cell suspension and vortexed gently. Gating was done using unstained cells (without PI) and positive control cells were stained with PI. The percentages of dead cells (PI-positive cells) were determined by using BD Accuri C6 flow cytometer (BD Biosciences, Wokingham, UK).
Cell Cycle Analysis
Cells (0.2x106) were plated in a 6-well plate with DMEM medium. After 48h of plating, cells were collected by trypsinization into a 15 ml tube. Cells were pelleted down by centrifugation at 1000 rpm, 4oC for 5 min, media was discarded and cells were re-suspended in 1 ml of ice cold PBS. Cells were then washed twice with 1 ml of ice-cold PBS by centrifugation at 1000 rpm, for 5 min at 4oC, PBS was discarded, and the cells were re-suspended in ice-cold 70% ethanol and kept overnight at -20oC. The following day, cells were pelleted by centrifugation at 1000 rpm for 5 min at 4oC, ethanol was discarded. Then cells were washed twice by centrifugation (1000 rpm for 5 min at 4oC) with 1 ml of ice-cold PBS followed by re-suspension in 200 µl of RNase (20 µg/ml) containing PBS. Cells were transferred into a 1.5 ml Eppendorf tube. Then 5 µl of propidium iodide (PI, stock 0.5 mg/ml) was added into 200 µl of cell suspension and kept in a dark environment at least 30 min before analysis. Cell cycle analysis was carried out by using BD Accuri C6 flow cytometer (BD Biosciences, Wokingham, UK).
Assessment Of Protein Half-life
To determine the protein half-life, cells were plated in a 6-well plate and treated with cycloheximide (100 µg/ml) (Sigma Aldrich) for 0h, 4h, 8h, and 16h. After the indicated time point, whole cell lysates were prepared. Western blotting for whole cell lysates was performed to determine the expression of ER in control and XBP1 KO MCF7 cells.
Cell Synchronization Using Css
For experiments with estradiol treatment, cells were cultured in phenol red free DMEM and CSS. MCF7 WT, MCF7 Y537S, and MCF7 D538G cells were synchronized by growing in phenol red-free DMEM containing 5% CSS for three days. For control and XBP1 KO MCF7 cells, synchronization and estradiol treatment experiments were carried out using phenol-red free DMEM containing 3% CSS. After synchronization, cells were treated with either vehicle or 17-estradiol for the indicated time points.
The data was analysed using GraphPad Prism 5.01. Data is presented as mean ± SD from three independent experiments unless otherwise stated. Densitometry analysis was carried out using Image J software. The survival of breast cancer patients was determined using Kaplan-Meier analysis. Other analyses of datasets are indicated in the figure legend. P-value was determined using Student’s t-test between independent groups, results with p < 0.05 were considered statistically significant.