Identification of Novel Genes and Pathways Regulating SREBP Transcriptional Activity
Figure 1
(A) Schematic representation of the SREBP cleavage assay. (B) Activity of wild-type (WT) versus mutant (Mut) SRE promoter. HEK-293 cells were set up in a 96-well plate (in triplicate). After 24 hours, cells were transfected with either WT (open bars) or mutant (black bars) luciferase reporter constructs, along with renilla luciferase construct and the indicated plasmid /cDNA. Cells were grown for an additional 24 hours before performing the assay. (C) Effects of 25-hydroxy cholesterol (25-OH chol.) on SREBP signaling. The assay was carried out under varying 25-OH cholesterol concentrations (0.1–5 µg/ml) and for different incubation periods (6, 12, and 24 hours). 25-OH chol. was added to cells 1 day after transfecting with the reporter plasmids, SRE-luciferase and renilla luciferase. Maximum suppression of SRE-luciferase signals was observed after 24 hours of incubation with 25-OH chol (shown here). The effects of DP-SCAP under high 25-OH cholesterol levels are significantly higher at all concentrations (Student's t-Test, p<0.005). (D) Effects of known repressors, activators and high cholesterol (25OH chol., 1 µg/ml) on the SREBP signaling pathway. The assay was carried out as in B. Basal refers to pcDNA3 overexpression. SCAP and DP-SCAP significantly activate the assay in the absence (white bars) or presence (black bars) of 25-OH cholesterol (Student's t-Test, p<0.05). (B–D) Error bars indicate standard deviations (n = 3). Where not visible, error bars are smaller than symbols. The graphs are representative of at least 2 independent experiments.