Fig 1: Effect of small-molecule inhibitors in EAC cell lines(A) mRNA levels of NACK and Notch1 of clinical samples derived from surgically resected primary EAC. Tumor tissue is compared with its normal tissue. (B) High levels of NACK and activated Notch1 expression are both observed in chemo-naive esophageal adenocarcinoma samples from endoscopic ultrasound (EUS) biopsies compared with normal esophageal tissue. Scale bars, 100 μM. (C) Colony formation assays were performed in OE19 EAC cell lines under treatment of different concentrations of Z271-0326 every other day for 10 days. The colony number was quantified and compared with the control (DMSO). (D) CSL-dependent affinity pull-down indicates Z271-0326 (3 μM) inhibits NACK, Notch1ICD, and MAML1 NTC assembly in OE19 cell line. (E) mRNA levels of HES5, CCND1, or TBP gene expression of OE19 treated for 6 h with DMSO, DAPT, or Z271-0326 were analyzed and normalized to HPRT1. (F and G) ChIP assay on the HES5 and CCND1 promoter using the indicated antibodies (n = 3). Error bars are representative of three independent experiments, and values indicate the mean ± SD. p values ≤0.05 are considered statistically significant and indicated by an asterisk. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.