Fig. 2.

LYPLAL1-DT is transcriptionally modulated by FOXO1.(A) Relative expression level of LYPLAL1-DT in TNBC cell lines, non-TNBC cell lines, and MCF10A. (B) Venn diagram delineating the identification of potential transcription factors (TFs) regulating LYPLAL1-DT. (C) Binding probability between the eligible TFs and the promoter of LYPLAL1-DT calculated in the JASPAR database. (D) Expression level of FOXO1 in nontumor normal samples and TNBC samples in the TCGA-TNBC cohort. (E) Kaplan–Meier curve showing that breast cancer patients with low expression of FOXO1 attain an inferior overall survival. (F) Expression level of FOXO1 protein (top) and FOXO1 RNA (bottom) in TNBC cell lines and MCF10A. (G) Significant positive correlation between LYPLAL1-DT and FOXO1 expression levels in the TCGA-TNBC cohort. (H) Validation of silencing FOXO1 in MDA-MB-231 and BT-549 cells and overexpressing FOXO1 in MDA-MB-157 cells (top), in addition with the corresponding expression alterations of LYPLAL1-DT (bottom). (I) ChIP-seq from the ENCODE database verifying FOXO1 binding peaks on the promoter region of LYPLAL1-DT in HepG2 cells. (J) The binding motif of FOXO1 (left) and the predicted binding site of FOXO1 on the promoter region of LYPLAL1-DT as well as the designed fragments for the further ChIP analysis (right). (K) Validation of FOXO1 antibody utilized in IP assay (top) and RT-qPCR result for ChIP assay using antibodies against FOXO1 and IgG (bottom). (L) Confirmation of RT-qPCR products in ChIP assay using nuclear acid electrophoresis. (M) Promoter dual-luciferase reporter assay conducted by cotransfections of plasmid pEZX-FR01 inserting LYPLAL1-DT promoter and plasmid overexpressing FOXO1 (FOXO1), or siRNA knocking down FOXO1 (siFOXO1), or the corresponding control plasmid (Scramble) or nontarget siRNA (siCtrl), which unraveled that FOXO1 definitely bound with the promoter of LYPLAL1-DT. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.