<p> An siRNA screen identifies the GNAS locus as a driver in 20q amplified breast cancer</p><p>Isaac Garcia-Murillas et al</p><p>Supplementary Data</p><p>Supplementary tables</p><p>Supplementary table 1. Cell lines used in siRNA screen along with screening conditions</p><p>Supplementary table 2. Full data set from siRNA screen</p><p>Genes included in three chromosomal regions along with CBS smoothed arrayCGH copy number (log2 ratio), dichomotised amplification status (1=amplified), gene expression data (for genes expressed in at least 2 cell lines), correlation between gene expression and copy number with Pearson Correlation coefficient, siRNA Z scores, percentage of amplified and non-amplified cancers with reductions in survival Amplicon driver screen identifying GNAS 2 with siRNA (Z score <-2), correlation between siRNA Z score and copy number or gene expression with Pearson’s Correlation coefficient</p><p>Supplementary table 3. Hits from screen</p><p> siRNA that reduced survival in a greater proportion of amplified cell lines than non- amplified cell lines (an siRNA reduced survival in an individual cell line with Z score</p><p><-2). Top shows hits which reduced growth in a maximum of 20% non-amplified cell lines. Bottom shows hits which reduced growth in >20% non-amplified cell lines, and therefore are at lower confidence of being amplicon selective. Included is the median level of gene expression in amplified and non-amplified cell lines, Student’s T test p value, and results from revalidation (Y – revalidated and amplicon specific, N – failed technical revalidation or not amplicon specific, blank – not examined in revalidation Amplicon driver screen identifying GNAS 3 screen). NA - Student’s T test p value not assessable as only a single cell line was amplified at that locus.</p><p>Supplementary table 4. Revalidation of screen hits</p><p>Twenty breast cancer cell lines (14 from screen and an additional 6 cell lines) were transfected with siRNA SMARTpools targeting the indicated genes, and survival was assessed after 4-6 days and expressed as a surviving fraction relative to growth in siCON transfected wells. The surviving fraction of cell lines with gene amplification was compared to those cell lines without gene amplification using Student’s t Test.</p><p>GNAS, EYA2, and C11orf67 were amplicon selective p<0.05. The data for GNAS and EYA2 is shown in Figure 2b.</p><p>Supplementary table 5. Pathological features of the small series of high grade</p><p>Oestrogen receptor positive HER2 negative breast cancers examined for</p><p>GNAS-XLs expression.</p><p>ER- oestrogen receptor, PR – progesterone receptor, HER2 – amplification status of</p><p>HER2, Grade – histological grade.</p><p>Supplementary figure legends Amplicon driver screen identifying GNAS 4</p><p>Supplementary figure 1 Analysis of GNAS</p><p>(a) Basal cAMP levels in GNAS over-expressing CAL120 cells. CAL120 cells infected with lentiviral pLEX empty vector, pLEX-Gs, or pLEX-XLs expression constructs, with two independent stable pools for each GNAS expression vector, and cAMP levels were assessed. * p<0.05 compared with pLEX levels, Student’s T test.</p><p>(b) Lysates were made form CAL120 cells transfected 24 hours earlier with multiple different siRNA targeting GNAS. Lysates were probed with indicated antibodies.</p><p>ERK1/2 phosphorylation is decreased in all GNAS siRNA transfected cells compared to siCON non-targeting siRNA.</p><p>(c) Publically available pooled shRNA data from an independent set of cell lines.</p><p>Displayed is the mean log fold change in abundance of shRNA targeting GNAS</p><p>(Cheung et al 2011). Left panel: Cancer cell lines with high-level amplification defined as >=1 relative copy number of the GNAS locus are more sensitive to GNAS shRNA</p><p>(p=0.004, Student’s T test). Right panel: Same data showing amplification of the</p><p>GNAS locus with a >=0.75 relative copy number (p=0.005, Student’s T test). For both panels the name of amplified cell lines are displayed.</p><p>Supplementary Figure 2. Oxerexpression of GNAS variants in MCF10A</p><p>Stable lentiviral pools were created in MCF10A cells with indicated lentiviral expression constructs. Left Immunofluorescent images of indicated cell lines pools with actin (green), E-cadherin (red), and DAPI (blue). Right Western blot of stable cell lines probed for Gs and XLs.</p>