adner. Purity and identity of this compound was verified by mass spectrometry and matched published standards. siRNA experiments were performed by transfecting MCF10A cells with Fostamatinib R788 siLentfect and 10 nM siRNA. c-MYC siRNA SMARTPool sequences : 5-CGAUGUUGUUUCUGUGGAA, 5-AACGUUAGCUUCACCAACA, 5-GAACACACAACGUCUUGGA, 5-ACGGAACUCUUGUGCGUAA, Luciferase: 5-UCGAAGUAUUCCGCGUACG. The previously validated shRNA targeting mTOR was obtained by cloning oligos into pLKO.1 and verified by sequencing 41. Barcoded vectors and generation of isogenic cell lines The stuffer fragment in the lentiviral vector pLKO.1 42 was replaced with a short linker sequence and barcodes flanked by primer sites and inserted 5 of the U6 promoter. This vector was then used to introduce stable DNA barcodes into cells by lentiviral transduction.
Cloning oligos into pLKO.2 using the AgeI and EcoRI restriction sites generated short hairpin RNA expressing vectors. GSK1120212 871700-17-3 An overview of all vectors used in the screen is provided in Supplementary Table 1. MCF10A isogenic cell lines overexpressing cDNAs or shRNAs were produced by lenti- or retroviral transduction and selection. Stable lines were cultured for approximately 4 weeks prior to the screen and barcoded by a second infection, when applicable. Prior to siRNA SMARTPool transfections MCF10A were infected with barcoded lentivirus. Screen set-up and Luminex assay For each compound a 4-point dose-response curve was determined in MCF10A cells using the Celltiter Glo assay. From these data, concentrations were selected for the Muellner et al. Page 7 Nat Chem Biol.
Author manuscript, available in PMC 2012 May 1. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript screen. All barcoded cell lines were pooled, counted and seeded in multiwell plates in quadruplicate. Compound or DMSO was added 16 h after seeding using a liquid handling robot. Medium was refreshed every second day and cells were cultured for a total of 9 days after which genomic DNA was isolated and barcodes were amplified. Genomic DNA extraction was performed with a liquid handler using the Genfind v2.0 kit. In brief, medium was removed and cells were washed twice with PBS. After lysis , 100 l raw lysate was transferred into 96-deepwell plates and 60 l Agencourt binding buffer was added. Beads were washed six times with 70% ethanol and purified genomic DNA was eluted in dH2O.
Barcodes were amplified in a 2-step protocol by PCR and linear amplification was performed with a 5 biotinylated primer. The single stranded product was hybridized to pre-coupled Luminex xMAP beads for 1.5 h at 40 in 384 well plates and streptavidin coupled phycoerythrin was added for 30 min. at 40. Finally, beads were washed once and samples were measured in a Flexmap 3D plate reader at 40. Quantitative real-time PCR RNA was isolated from sub-confluent cells using Trizol. After purification and DNase treatment reverse transcription was performed using random hexamer primers and RevertAid reverse transcriptase. Quantitative real-time PCR was carried out using the iTaq SYBR Green Supermix according to the manufacturer,s instructions. Measurements were performed in triplicate and related to GAPDH as a reference gene.
All primer sequences are listed in Supplementary Table 6. GFP competition assay Cells were infected with vectors carrying the cDNAs for ICN1 and GFP or an empty control vector. After infection, cells were pooled and distributed among multiple 6-well plates for BEZ-235 or DMSO treatment. GFP positive cells were measured by FACS or microscopy. For the microscopy analysis, 10 randomly chosen fields were imaged for each cell line-drug combination and cells were quantified using CellProfiler. Uninfected cells were used to determine background fluoresce