Fig. 6

FOXP1 acts as a transcription factor of PLOD2 in CAAs. a FOXP1 was selected as the potential transcription factor of PLOD2 based on a series of databases and qPCR verification. First, the promoter sequence of PLOD2 was obtained via the Ensemble project and Mapviewer. Then, 769 potential transcription factors were predicted using the Genomatix database, and eight of the transcription factors with highest potential were screened out based on the prediction score (the matrix sim). Finally, based on qPCR analysis and inhibitor treatment, FOXP1 was identified as a potential transcription factor. b The gene expression of eight potential transcription factors were evaluated in adipocytes cultivated in the presence or in the absence of tumor cells (MDA-MB-231 and SKBR-3 cells) for 72 h. c The expression of FOXP1 and FOXL1 were evaluated in adipocytes cultivated in the presence or in the absence of tumor cells (MDA-MB-231 and SKBR-3 cells) with tumor cells in the presence of tiplaxitin for 72 h. d,e The PI3K/AKT signaling pathway inhibitor (LY294002) and tiplaxitin inhibited the nuclear translocation of FOXP1 through the separation of the cytoplasm and nuclear proteins of adipocytes cultivated in the presence or in the absence of tumor cells (MDA-MB-231 and SKBR-3 cells) for 72 h. f The PI3K/AKT signaling pathway inhibitor (LY294002) inhibited the nuclear translocation of FOXP1 through the separation of the cytoplasm and nuclear proteins of adipocytes cultivated in the presence or in the absence of PAI-1 (200 ng/ml). g Adipocytes grown on coverlips cultured in the presence or in the absence of tumor cells with tumor cells in the presence of tiplaxitin or LY294002 for 72 h. Cells were fixed and stained with the FOXP1 antibody (green), nuclei were stained with Hochest (blue). Scale bars, 100 μm. h Three putative bind regions of FOXP1 upstream of the transcription start site (TSS) of PLOD2 gene were found using ChIP assay. The PI3K/AKT signaling pathway inhibitor (LY294002) was used to inhibit the binding regions of the PLOD2 gene. Precipitated DNA fragments in ChIP assays were examined using qPCR. Immunogobulin G (IgG) was used as a negative control, schematic representation of FOXP1-binding sites upstream of the TSS of PLOD2 using ChIP assay. The P-values < 0.05 was considered statistically significant for all tests