Figure 4: Activation of LXRs regulates expression of kinases, phosphatases and reduces PI3K signalling. (A) MCF7 cells were treated with DMSO or 5 μM GW3965 for 16 h. Relative mRNA expression levels of the key LXR target gene (ABCA1) and genes involved in the control of AKT signalling pathway (AKT1, AKT2, PHLPP, PHLPPL, and PTEN) were quantified by qPCR. (B) Cell lysates from MCF7 cells were isolated and protein expression analysed by Western blotting for phosphatase (PTEN, PHLPP, and PHLPPL). β-actin was used as loading control. Relative density of the protein's band was quantified as described in material and methods. (C) MCF7 and SW480 cells were cultured with 1% serum in growth medium and treated with DMSO or 5 μM GW3965 for 16 h. Cells were activated with IGF-1 (50ng/ml) for 20 min, whole cell extract isolated and Western immunoblotting performed with various antibodies targeting PI3K. GAPDH was used as an internal control demonstrating equal protein loading. (D) Inhibition of class IA phosphoinositide 3-kinase (PI3K) activity with 5 μM GW3965 for 16 h measured as phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated from immunoprecipitated PI3K in a kinase assay using a competitive ELISA method. The data are presented as mean ± SEM. *P< 0.05, **P< 0.01, and ***P< 0.001 versus vehicle by Student's t-test.