Research Paper Volume 16, Issue 11 pp 9410—9436

Figure 4. SREBP1 activation is necessary for APOA5 transcriptional regulation in PIK3CA mutant CRC cells. (A) The protein levels of Akt, p-Akt and SREBP1 were measured by Western blot assays in HCT-116 cells transfected with PIK3CA-E545K or vector plasmids. (B) Immunoblotting assays were performed with nuclear and cytoplasmic extracts from HCT116/Vector and HCT116/E545K cells, which showed increased mature SREBP1 (mSREBP1, 65 kD) and decreased full-length SREBP1 (flSREBP, 125 kD) in HCT116/E545K cells. (C) Transfected cells were cultured in the presence or absence of 1 mg/ml 25-hydroxycholesterol (sterols) for 2 hours, and nuclear extracts were analyzed for expression of mSREBP1. (D) Whole cell lysates of sterols treated cells were analyzed for expression of APOA5 and p-Akt. (E) ChIP-PCR assays were performed to access the binding between SREBP1 and APOA5 promoter. (F) SREBP1 knockdown was performed with siRNA transfection. APOA5 protein levels were quantified 72 hours after cell infection. (G) The mRNA levels of SREBP1 and APOA5 were quantified by real time PCR. (H) Transfected HCT116 cells were further infected with luciferase (LUC) reporter constructs which contains a wild type or mutant APOA5 promoter fragment as shown. Luciferase activity was measured and compared between different groups. Results are representative of at least three independent experiments. p-values < 0.05 are represented as a^*.