ATF4 increased the transcriptional activity of ILEI to promote EMT in ESCC. A. ATF4 protein expression in fractionated cells with stably expressed Sh-TMTC3 and Sh-NC (left). The quantification of ATF4 intensity in WB (right). Nuc, nuclear. Cyto, cytoplasm. β-tubulin is a cytoplasmic control, LaminB1 is a nuclear control. B. IF images for the localization of ATF4 in TMTC3 cells transfected with siRNA and negative control. Scale bar, 30 µm. C. Binding of ATF4 to the ILEI promoter region in KYSE410 and KYSE450 cells was analyzed by ChIP-qPCR. IgG was used as a nonspecific binding control. **, p < 0.01. ***, p < 0.001. D. The relative luciferase reporter activity of ATF4 on the ILEI promoter in KYSE180 and KYSE450 cells. ***, p < 0.001. n.s., no significance. E. The relative dual-luciferase activity of the ILEI promoter in TMTC3 cells with stable knockdown. ***, p < 0.001. F. Model of the ATF4-ILEI axis in regulating EMT in SCCs.