Methods DNA constructs Constitutively active Notch constructs were made with cDNA encoding either membrane tethered, Drosophila selleck chem Notch, constitutively activated by the removal of the extracellular domain, or the soluble intracellular domain. These truncated Notch constructs were cloned into the pIZ V5 His expression vector producing non tagged proteins. The control expression plasmid was constructed by cloning firefly luciferase into the same pIZ V5 His expression vector. The luciferase reporter construct con tains a 1. 4 kb tandem duplication of E m3 upstream regulatory sequences, cloned into a pGL2 Basic vector, as described. Genome wide RNAi method A total of 23,560 dsRNAs, made available from the Dro sophila RNAi Screening Center at Harvard Medical School, were screened by the following method, Kc167 cells were washed three times and resuspended in serum free Sangs M3 medium at a concentration of 5 �� 105 cells ml.
Using a robotic liquid handler, 104 cells were uniformly dispensed into the wells of 384 well polypropylene plates containing dsRNA and incubated for 45 min at room temperature. An equal volume of M3 medium containing 10% fetal bovine serum was added and incubated for four days. On day four, the RNAi treated cells were diluted with 100 ul of medium, mixed and 20 ul were dispensed into the wells of six new 384 well plates, pre aliquoted with 20 ul of transfection mix. The six plates contained the three different transfection mixes, each in duplicate. Transfection mixes were prepared with Effectene Trans fection Reagent, following the manufacturers guidelines.
Luciferase activity was measured 24 h post transfection using the Steady Glo Luciferase Assay Sys tem. This method requires only two plasmids to be transfected at one time and gave acceptable signal to noise ratios for high throughput screening in 384 well plate format. Whereas, the conven tional renilla dual glo assay was not robust enough to scale to 384 well format with this Notch reporter sys tem using an endogenous target. In contrast to path ways with soluble ligands, the reporter and constitutively active Notch constructs are required to transfect the same cell to activate transcription. With the renilla dual glo system, adding the control con struct required the co transfection of three individual plasmids and this reduced the signal to noise ratio to insufficient levels.
Data analysis Duplicate measurements for each of the three signals were averaged, Notch specific E m3 promoter in the presence of activated Notch, the E m3 promoter alone and the unrelated viral promoter OplE2. The Necn m3 luc signal was Dacomitinib normalized two different ways, by either the m3 luc or con luc signals. The z scores of the log2 ratios were calculated by using the standard deviation and mean of the measurements that corresponded to the 96 wells of the original dsRNA stock plates.