Cell lines, compounds, plasmids and siRNA
MCF7, CAL51, HeLa, A549, NCI-H226, PC3, DU145 and MCF10A cells were obtained from ATCC (USA) and maintained according to the supplier's instructions. WEE1 inhibitor (681637) was obtained from Calbiochem (UK). MCF7 and HeLa cells were transfected with SMARTpool siRNAs using Dharmafect 3 transfection reagent; A549 and NCI-H226 cells were transfected with SMARTpool siRNAs using Dharmafect 1 transfection reagent according to manufacturer's instructions (Dharmacon). CAL51 cells were transfected with SMARTpool siRNAs using Oligofectamine transfection reagent according to manufacturer's instructions (Invitrogen). DU145 cells were transfected with SMARTpool siRNAs using Lipofectamine 2000 transfection reagent according to manufacturer's instructions (Invitrogen). The kinase siRNA library (siARRAY – targeting 779 known and putative human protein kinase genes) was obtained in ten 96 well plates from Dharmacon (USA). Each well in this library contained a SMARTpool of four distinct siRNA species targeting different sequences of the target transcript. Each plate was supplemented with siCONTROL (ten wells, Dharmacon (USA)). The WEE1 ONTARGETplus SMARTpool and ONTARGETplus siControl were obtained from Dharmacon (USA).
Antibodies targeting the following epitopes were used: WEE1 (4936, Cell Signaling, UK), PARP (9542, Cell Signaling, UK) and β-tubulin (T4026, Sigma, UK). All secondary antibodies used for western blot analysis were HRP conjugated.
siRNA screen method
Cells plated in 96 well plates were transfected 24 hours later with siRNA (final concentration 100 nM), as per manufacturer's instructions. Each siRNA plate was supplemented with 10 wells of siControl. Twenty four hours following transfection, cells were trypsinised and divided into three identical replica plates. Media was replenished after 48 hours and 96 hours, and cell viability was assessed after seven days using CellTiter Glo Luminescent Cell Viability Assay (Promega, USA) as per manufacturer's instructions. Data from each cell line was processed as follows: the luminescence reading for each well on a plate was log2
transformed and expressed relative to the median luminescence value of all wells on the same plate (plate centering). This data was then normalised according to the median of the entire screen data, using the median absolute deviation (MAD) to estimate the true variation within each screen 
. This normalisation represented the effect of each SMARTpool in each cell line as a Z score 
and allowed the effects of each SMARTpool on viability to be compared across the cell line panel. A Z score≤−3 was taken as the significance threshold for reduced cell viability, representing three MADs from the median and approximating to three standard deviations.
RNA was extracted from cell lines with Trizol and phenol/chloroform extraction followed by isopropanol precipitation. For reach cell line, triplicate extractions and profiles were performed. Biotin-labeled cRNA was produced by means of a linear amplification kit (IL1791; Ambion, Austin, TX, http://www.ambion.com
) using 250 ng of quality-checked total RNA as input. Chip hybridisations, washing, Cy3-streptavidin (Amersham Biosciences) staining, and scanning were performed on an Illumina BeadStation 500 (San Diego, http://www.illumina.com
) platform using reagents and following protocols supplied by the manufacturer. cRNA samples were hybridised on Illumina human-6 v2 BeadChips, covering approximately 47,000 RefSeq transcripts. The random distribution of large populations of oligonucleotide-coated beads across the available positions within the human-6 v2 chip enables, on average, 30 intensity measurements per RefSeq, yielding quantitative assessments of gene expression 
. All basic expression data analysis was carried out using the manufacturer's software BeadStudio 3.1. Illumina expression profiles were performed in triplicate, the raw data were then variance-stabilizing transformed and robust spline normalised using the lumi package in the Bioconductor software 
. Expression values for each sample were median scaled and the mean expression value was established over the three replicates. Genes with significant difference in expression between cell lines were identified by one-way analysis of variance (ANOVA). This transcript profiling data is now publicly available (ArrayExpress accession: E-TABM-610).
Correlation of siRNA Z score with gene expression
The correlation between siRNA Z score and normalised gene expression was examined for genes where siRNA caused significant loss of viability (Z<−3). Z score was compared to normalised gene expression using Pearson correlation coefficient. A gene was taken as being significantly correlated if the Pearson correlation coefficient was significantly different to the null hypothesis, the correlation was inverse, and the variation in gene expression between cells lines were significantly different as assessed by one-way ANOVA.
Array CGH analysis method
Genomic DNA was extracted from cell lines using the QIAamp DNA Blood Mini Kit (51104, Qiagen), according to manufacturer's instructions. Microarray-based CGH analysis was performed on an in-house 32K tiling path BAC array platform as previously described 
. For copy number correlations, the average of adaptive weight smoothed (AWS) ratios of BACs containing the gene of interest were used for copy number correlations, and copy number assigned as previously described 
. Briefly, AWS smoothed log2 ratio values <−0.12 were categorised as losses, those >0.12 as gains, and those in between as unchanged. Amplifications were defined as smoothed log2 ratio values >0.4 
. Data processing and analysis were carried out in R 2.0.1 (http://www.r-project.org/
) and BioConductor 1.5 (http://www.bioconductor.org/
), making extensive use of modified versions of the packages aCGH, marray and aws in particular.
Cell viability assay to measure WEE1 siRNA sensitivity
Cells plated in 96 well plates were transfected 24 hours later with WEE1 ONTARGETplus SMARTpool or ONTARGETplus siControl (final concentration 100 nM), as per manufacturer's instructions. Twenty four hours following transfection, cells were trypsinised and divided into three identical replica plates. Media was replenished after 48 hours and 96 hours, and cell viability was assessed after seven days using CellTiter Glo Luminescent Cell Viability Assay (Promega, USA) and expressed relative to mean luminescence in the wells transfected with siControl.
Cell viability assay to measure drug sensitivity
Cells were plated in 96 well plates and exposed to various doses of WEE1 inhibitor (Calbiochem, Cat. No. 681637, 4-(2-Phenyl)-9-hydroxypyrrolo[3,4-c]carbazole-1,3-(2H,6H)-dione (PHCD)) 
. Cell viability was assessed by CellTiter Glo Luminescent Cell Viability Assay (Promega, USA) 48 hours later and surviving fraction for each dose of drug assessed by dividing the luminescence value of drug treated by the luminescence value of vehicle.
Protein lysates were prepared using RIPA lysis buffer (50 nM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.1% DOC, 1% TritonX-100, 50 mM NaF, 1 mM Na3VO4 and protease inhibitors). 100 µg of total cell lysate was loaded onto prefabricated 4–12% Bis-Tris gels (Invitrogen), with full range rainbow molecular weight marker (GE Healthcare, UK) as a size reference, and resolved by SDS-PAGE electrophoresis. Proteins were transferred to nitrocellulose membrane (Bio-rad, USA), blocked and probed with primary antibody diluted 1 in 1000 in 1×TBS-T with 5% BSA overnight at 4°C. Secondary antibodies were diluted 1 in 5000 in 1×TBS-T with 5% skim milk and incubated for one hour at room temperature. Protein bands were visualised using ECL (GE Healthcare, UK) and MR or XAR film (Kodak).
Validation of gene silencing by siRNA
Validation of RNAi gene silencing was determined by western blotting. Cells were transfected with WEE1 ONTARGETplus SMARTpool or ONTARGETplus siControl, and protein lysates were made 48 hours later and western blotted for WEE1 expression with β-tubulin as a loading control.
PARP cleavage western blotting
Cells were transfected with WEE1 ONTARGETplus SMARTpool or ONTARGETplus siControl, and total cell lysates were made 48 hours later and western blotted for PARP with β-tubulin as a loading control. Cells were treated with 5 µM Wee1 inhibitor for 0, 6, 24 and 48 hours. Total cell lysates were made at the time points and western blotted for PARP with β-tubulin as a loading control.
Caspase 3,7 activation assay
Cells were transfected with WEE1 ONTARGETplus SMARTpool or ONTARGETplus siControl, and caspase 3,7 activation was measured 48 hours later using Caspase-Glo 3/7 Assay (G8091, Promega) and expressed relative to mean luminescence in the wells transfected with siControl. Cells were treated with 5 µM Wee1 inhibitor and caspase 3,7 activation was measured 24 hours later using Caspase-Glo 3/7 Assay (G8091, Promega) and expressed relative to mean luminescence in the wells treated with vehicle.
WEE1 immunohistochemical staining
Immunohistochemistry for WEE1 was performed with a rabbit polyclonal antibody (Cell Signalling; 4936) at a dilution of 1/20 and developed with the dual Envision kit (Dako®, Glostrup, Denmark). Details of this cohort of patients are described elsewhere 
. Antigen retrieval was performed at 98°C for 30 minutes in citrate buffer pH 6 (Labvision) in the Labvision pre-treatment module. WEE1 immunohistochemical distribution on tissue microarray sections was analysed by two of the authors (JR-F and KS) on a multi-headed microscope. Only nuclear reactivity was considered specific. Cases were scored according to the Allred scoring system 
and a cut off of >5 (median score in the series) was adopted. The analysis was performed blinded to the results of other immunohistochemical markers and patients' outcome. All cases were classified into luminal, HER2 and basal-like groups according to the immunohistochemical panel described by Nielsen et al.