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. 2021 Feb 25;11:605025. doi: 10.3389/fonc.2021.605025

Figure 3.

Figure 3

FOXP2 stimulated the expression of PHF2 during MET of breast cancer cells. (A, B) The expression levels of PHF2 and FOXP2 were upregulated during CTx-induced MET of MDA-MB-468 breast cancer cells. MDA-MB-468 cells were treated with different concentrations of CTx (50 or 100 ng/ml) for 7 days. The levels of FOXP2, PHF2, E-cadherin, and Vimentin were examined by Western blotting (A) and qPCR (B), respectively. (C) The overexpression of FOXP2 stimulated PHF2 expression in MDA-MB-468 cells. MDA-MB-468 cells were transfected with pHis-FOXP2 or pCMV-EGFP. The levels of FOXP2 and PHF2 were examined by Western blotting and qPCR. (D) FOXP2 bound to the endogenous promoter of PHF2. Gene sequence analysis was performed to predict positions of putative FOXP2 binding sites in -2 kb human PHF2 promoter and the primers for ChIP assays were designed. The chromatin of MCF-7 cells was cross-linked, sonicated, and immunoprecipitated (IP) with either FOXP2 antibody or rabbit IgG. The amount of promoter DNA associated with the IP chromatin was measured by qPCR with primers specific to PHF2 promoter regions -1,838 bp to -1,693 bp and -1,519 bp to -1,403 bp. (E) FOXP2 bound to PHF2 promoter region -1,765 bp to -1,743 bp. The nuclear extracts were prepared from pHis-FOXP2-transfected cells and used for EMSAs with a FAM-labeled DNA probe synthesized from PHF2 promoter sequence -1,765 bp to -1,743 bp (PHF2 pro). The unlabeled probe (50×) or 1 μg of FOXP2 antibody (α-FOXP2) was added to the reaction to show the specificity of FOXP2/DNA complex formation. EMSAs with a FAM-labeled mutated probe were also performed (Mut pro). (F) The PHF2 promoter was activated by FOXP2 in breast cancer cells. A luciferase reporter plasmid (1.5 μg) containing the fragment of -2,000 bp to +60 bp of PHF2 promoter and loading control pRL-CMV luciferase reporter plasmid (20 ng) was transfected into MDA-MB-468 cells with different amounts of pHis-FOXP2 (0, 0.5, 1.0, and 1.5 μg, balanced with the different amounts of empty expression vector). Protein lysates were prepared at 48 h following transfection and then used to measure dual luciferase enzyme activities. The asterisks indicate statistically significant changes: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.