Fig 2: E2F1 regulates FAT1 transcription. (A) Putative binding sites of E2F1 on the FAT1 promoter region from different cohorts in encyclopedia of DNA elements (ENCODE) database. Red box represents binding reads of E2F1; (B) Enrichment of E2F1 on FAT1 promoter in KYSE30 cells shown by chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR). Enrichment is determined as the amount of FAT1 promoter associated to E2F1 relative to immunoglobulin G (IgG) control; (C) Luciferase activity of pGL3-FAT1 vector was measured in KYSE30 cells upon E2F1 knockdown. Data are presented as ratio of the firefly luciferase activity to Renilla luciferase activity; RT-qPCR (D) and Western blot (E) analyses of E2F1 expression in KYSE30 cells upon E2F1 knockdown. Relative RNA levels of E2F1 were normalized to endogenous GAPDH; (F) RT-qPCR analysis of FAT1 expression in KYSE30 cells as described in (D). Relative RNA levels of FAT1 were normalized to endogenous GAPDH. *, P<0.05; **, P<0.01; ***, P<0.001vs. control.

Fig 3: Verification of the knockdown efficiency of FAT1 in YSE2 and Colo680N cells. (A) The mRNA expression pattern of FAT1 in non-transformed esophageal epithelial SHEE cells and 8 ESCC cell lines as detected by qPCR (upper panel) and RT-PCR (Lower panel). (B) Upper panel: FAT1 protein level in non-transformed esophageal epithelial SHEE cells and 8 ESCC cell lines as detected by in-cell western assay. Channel 700 represents cell number and channel 800 represents FAT1. The merge represents the protein expression of FAT1 in these cells. Lower panel: Quantification of the protein level of FAT1 in various ESCC cell lines. (C) Examination of the transfection efficiency of the FAT1shRNA plasmid into the YSE2 and Colo680N cells by fluorescence microscopy (scale bar, 100 µm). (D) Knockdown efficiency of FAT1 in YSE2 and Colo680N cells was verified by qPCR (upper panel) and RT-PCR (lower panel). (E) Knockdown efficiency of FAT1 in YSE2 (left panel) and Colo680N cells (right panel) was verified in-cell western assay. Channel 700 represents cell number and channel 800 represents FAT1. The merge represents the protein expression of FAT1 in these cells. All experiments were repeated three times independently. FAT1, FAT atypical cadherin 1.

Fig 4: FAT1 mutations frequently occur in ESCC and other squamous cell carcinomas, and FAT1 exhibits downregulated expression in ESCC. (A) The frequency of mutations of FAT1 in four types of squamous cell carcinomas (ESCC, esophageal squamous cell carcinoma; HNSCC, head and neck squamous cell carcinoma; LSCC, lung squamous cell carcinoma; OSCC, oral squamous cell carcinoma) in the TCGA database. (B) Overall and representative images of FAT1 expression in tumor tissues (T) and adjacent non-tumor tissues (N) from paraffin-embedded formalin-fixed ESCC tissue microarrays containing 76 tumors and corresponding non-tumor tissues by IHC. Left panel: magnification ×10 (scale bar, 4 µm); right upper panel: magnification ×200 (scale bar, 100 µm); right lower panel: statistical analysis of FAT1 protein level based on TMA data. FAT1, FAT atypical cadherin 1.

Fig 5: Downregulation of FAT1 impairs migration ability of OSCC. Knockdown by siFAT1-1 and siFAT1-2 inhibited HN6 and HN30 cells migration via transwell chambers without Matrigel (A) and wound healing migration assays (B). **P < 0.01; ***P < 0.001; ****P < 0.0001.