Figure 7

AURKA negatively regulates FOXP1-mediated FBXL7 promoter activity. (a) AURKA-interacting transcription factor-like proteins were identified using in vitro GST pulldown (red area). FBXL7 promoter-binding proteins were deduced from MatInspector analysis (green area). FOXP1 was present in both data sets and selected for further studies. (b) HEK293T cells were transfected with His-tagged FOXP1 and AURKA plasmids and whole-cell lysates co-immunoprecipitated with control IgG, His and AURKA antibodies. Input (1/25 of IP) and immunoprecipitates were processed for immunoblotting with the indicated antibodies. (c) In silico protein–protein interaction between AURKA and FOXP1. PDB files were obtained from PDB databank (http://www.rcsb.org/pdb) and analyzed for AURKA and FOXP1 interacting residues using PRISM webserver (cosbi.ku.edu.tr/prism/). (d) Recruitment of FOXP1 and AURKA to FBXL7 promoter in gastric cancer cells. AURKA and FOXP1 occupancy at the FBXL7 promoter was analyzed by chromatin immunoprecipitation (ChIP) using AURKA, FOXP1 and control IgG antibodies. Input and ChIP DNA was processed for conventional PCR using three different primer pairs, spanning a 2 kb region of FBXL7 promoter. (e) Validation of FBXL7 promoter as a direct target of FOXP1. HEK293T cells were transfected with empty pGL3 basic or pGL3-FBXL7 promoter and pcDNA vector, AURKA and FOXP1 as activators. After 24 h, cells were harvested and processed for dual luciferase activity. FBXL7 promoter activity in response to pcDNA vector was set 100% and compared with other samples. Each sample was transfected in triplicates and three repeated experiments were performed in which similar results were obtained. (f) The luciferase activities of FBXL7 promoter 5′ deletion constructs in response to pcDNA vector or FOXP1. The −1500 bp FBXL7 promoter activity was set as 100% and compared with −1000, −500 and −212 bp deletion constructs. FOXP1 strongly activated all 5′ deletion constructs affirming that FBXL7 promoter has multiple FOXP1-binding sites. Each sample was transfected in triplicates and three independent experiments were performed in which similar results were obtained. Data present mean±s.e.m. of three independent experiments and statistical analysis was performed with one-way ANOVA, *P⩽0.05; **P⩽0.01; ***P⩽0.001.