ion_Platelet-endothelium-leucocyte interactions Serine protease n-3,6 Polyunsaturated & unsaturated fatty acid biosynthesis Arachidonic acid production G-protein, small GTPase & RAS-related GTPase Neuroendocrine tumors Lipid & sphingolipid metabolism Autoimmune diseases Cell structure & Gap junction Gene from biological analysis SREBF1, INSIG2, ACSL4 SCARF1 LACTB, ABHD4, MASP2 ACSL4 ACSL4 RAB20, RAB8A, RAP2B BIN1, RAB8A SPTLC1, PLCB1 MBP, SPTLC1 CSNK1E, DGCR14, MBP Novel 16483784 genes regulating SREBP signaling that were identified in the biological rescreens were mapped onto the pathway analysis data. Shown here are the novel genes that showed up in both biological as well as pathway analyses. doi:10.1371/journal.pone.Clemizole hydrochloride 0005197.t004 Genome-wide cDNA study and luciferase assays A reverse transfection protocol was followed for testing the 10,000 genes for their effect in the SREBP signaling assay. Trypsinized HEK-293 cells were added to 384-well white opaque bottom plates, containing the cDNA clone and transfection mix, at density of 2500 cells/well at 25 ml per well using a Multidrop 384 and incubated at 37uC in 5% CO2. The transfection mix consisted of 17.5 ng reporter plasmid/ well, 0.7 ng Renilla/well and Fugene 6 at a ratio of 3 ml Fugene to 1 mg of DNA in 5 ml of Optimem. The transfection mixture was added to the 384-well plates containing the cDNA clone using a FlexDrop. The cDNA’s were screened at a concentration of 120 ng/well. For 22431203 cholesterol stimulation, 25-hydroxycholesterol in 0.01% ethanol was added to cells at a concentration of 1 mg/ml, and incubated for 24 hours. Firefly and Renilla luciferase activity was measured 40 hours post transfection using the Dual Glow assay system. Plates were allowed to cool for 10 minutes before 30 ml of each assay reagent was added. The plates were shaken for 10 minutes on a multi-plate shaker. Luminescence was determined using an EnVision plate reader with a 100 msec integration time. For luciferase assays carried out in 96-well plates, all reagents were proportionally increased 4-fold. Supporting Information Data analysis and 2D-normalization of genome-wide study Results were analyzed using Spotfire Decisionsite software and Microsoft Excel. For the genome-wide study, luciferase ratios were normalized as follows. The firefly-luciferase readout values were first normalized using the corresponding renilla-luciferase readout values. The firefly-renilla ratio was further normalized as follows. The one-dimensional values were obtained by scaling the ratios with the plate median in order to remove plate-to-plate variation. The two-dimensional values were obtained by further removing the well-to-well variations through an iterative procedure. Finally, the normalized values were standardized to obtain the NZ score, which is a more robust equivalent of the more commonly used Z score. If the distribution is perfectly normal, NZ score will be the same as the Z score. Complete list of validated activators of SREBP signaling. Found at: doi:10.1371/journal.pone.0005197.s002 SREBP Activity Modifiers regulators of gap junctions tended to inhibit SREBP activity, whereas negative regulators tended to activate SREBP. The most potent inhibitor and activator among these were validated in secondary assays. Some genes showed isoform selectivity 
for SREBP activity, e.g. the epsilon isoform of casein kinase 1 was a more potent activator than the delta and gamma isoforms. Nevertheless, the consistency of results across sever
