Fig 2: Knockdown of Forkhead box P2 (FOXP2) reverses the effect of miR-300 inhibitors on gastric cancer (GC) cells. (A,B) Knockdown of FOXP2 could restore the effect of miR-300 inhibitors on cell proliferation in BGC-823 and SGC-7901 cells by colony formation assays. (C,D) Knockdown of FOXP2 could restore the effect of miR-300 inhibitors on cell proliferation in BGC-823 and SGC-7901 cells by 5-ethynyl-2'-deoxyuridine (EdU) assays, scale bar = 100µm. (E) The promotion of cell apoptosis by miR-300 inhibitors was restored by knockdown of FOXP2 in BGC-823 and SGC-7901 cells. (F,G) The inhibition of cell migration and invasion by miR-300 inhibitors was restored by knockdown of FOXP2 in BGC-823 and SGC-7901 cells. (H,I) The effect of miR-300 inhibitors on autophagy can be recovered by knockdown of FOXP2 in BGC-823 and SGC-7901 cells, scale bar = 20µm. (J,K) Expression levels of apoptotic, epithelial-mesenchymal transition (EMT) and autophagy proteins were examined by western blotting in BGC-823 and SGC-7901 cells. (*p < .05, **p < .01, ***p < .001. Data are expressed as the means ± SDs)

Fig 3: Forkhead box P2 (FOXP2) transcriptionally inhibits MET and regulates autophagy through AKT/mTOR axis which regulates the malignant progression of gastric cancer (GC) cells. (A) Schematic diagram showed three luciferase reporters cover different DNA sequences of MET promoter region. (B) Luciferase reporter assay analysis of three MET promoter luciferase reporters in BGC-823 cells transfected with FOXP2 or pcDNA. (C) Schematic diagram showed three primers for ChIP assay. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in BGC-823 cell after pulling down FOXP2 using three pairs of primers to investigate the potential FOXP2 binding sites in MET promoter region. (E) Expression levels of FOXP2, MET/AKT/mTOR and autophagy proteins were examined by western blotting. (F,G) The effect of overexpression of circST3GAL6 on autophagy can be recovered by knockdown of FOXP2 via confocal microscopy (scale bar = 20µm) and transmission electron microscopy scale bar = 1µm in BGC-823 cell. (H,I) The effect of overexpression of FOXP2 on autophagy can be recovered by 3-methyladenine (3-MA; 5 nM, 24 h) via confocal microscopy (scale bar = 20µm) and transmission electron microscopy (scale bar = 1µm) in BGC-823 cell. (J) Expression levels of autophagy-related proteins were examined by western blotting in BGC-823 cell. (K–M) 3-MA (5 nM, 24 h) could restore the effect of FOXP2 overexpression on cell proliferation in BGC-823 cell by CCK8, colony formation and 5-ethynyl-2'-deoxyuridine (EdU) assays. (N) 3-MA (5 nM, 24 h) could restore the effect of FOXP2 overexpression on cell migration and invasion in BGC-823 cell transwell assays. (*p < .05, **p < .01, ***p < .001. Data are expressed as the means ± SDs)

Fig 4: Structural organization of FOXP2 and ß-catenin. (A) Disorder prediction using PONDR® (www.pondr.com) for FOXP2 indicating the a-helix around residue 270, zinc finger, leucine zipper, and the forkhead as ordered. (B) Schematic representation of FOXP2 constructs used for cell-based and in vitro experiments.

Fig 5: Expression of FOXPs in different human cancers. (A) Expression of FOXP1 in different tumor types in TCGA database was detected by TIMER. (B) Expression of FOXP2 in different tumor types in TCGA database was detected by TIMER. (C) Expression of FOXP3 in different tumor types in TCGA database was detected by TIMER. (D) Expression of FOXP4 in different tumor types in TCGA database was detected by TIMER. E, GEPIA was used to verify the difference of FOXP1 expression between thyroid cancer tissues and normal thyroid tissues in TCGA database. (F) GEPIA was used to verify the difference of FOXP2 expression between thyroid cancer tissues and normal thyroid tissues in TCGA database. (G) GEPIA was used to verify the difference of FOXP3 expression between thyroid cancer tissues and normal thyroid tissues in TCGA database. (H) GEPIA was used to verify the difference of FOXP4 expression between thyroid cancer tissues and normal thyroid tissues in TCGA database. *p < 0.05; **p < 0.01; ***p < 0.001.