Fig 2: The ZEB1–CtBP inhibitor NSC95397 evicts Ctbp from Zeb1 complex and thereby upregulates the miR-200 family to downregulate Zeb1 expression in mRMEVCs.Downregulation of Ctbp by lentiviral shRNA in mRMEVCs did not affect a expression and (b–c) relocation of Zeb1, and thereby (d) cell proliferation. e Co-immunoprecipitation of Zeb1 using a CtBP antibody against both CtBP1 and 2 (CtBP-IP) from total protein (Input) isolated from mRMVECs treated with either 10 µM NSC95397 or 10 mM MTOB and WB for Zeb1 and Ctbp. f Expression of the miR-200 family genes detected by qPCR in mRMVECs treated with 10 mM MTOB or 10 µM NSC95397. It appears that only 10 µM NSC95397 significantly reduced the alkali-induced corneal NV evaluated by criteria of (g) opacity, (h) NV score, and i vessel size. *p ≤ 0.05; **p ≤ 0.01. Scale bars represent 100 µm.

Fig 3: Survival curves of CtBP2 (A) or TGIF (B) expression in patients with ESCC. A total of 108 patients with follow-up data were evaluated and immunohistochemical staining followed by Kaplan-Meier survival analysis was performed. Patients with a low expression of CtBP2 or TGIF had a longer cumulative survival. CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor; ESCC, esophageal squamous cell carcinoma.

Fig 4: Signaling pathway involved in the biological activity of CtBP2 in ECA109 cells. (A) Viability of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 24, 48 and 72 h, was measured using CCK-8 assay. (B) The proliferation of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 48 h was measured using EdU staining. Scale bar, 50 µm. (C) Statistical analysis of EdU staining results. (D) The migration of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 48 h was measured using wound healing assay. Scale bar, 100 µm. (E) Statistical analysis of the wound healing assay results. (F) The migration and invasion of ECA109 cells transfected with LV-CtBP2 and treated with 10 µM XAV939 for 48 h was measured using Transwell assay. Scale bar, 100 µm. (G) Statistical analysis of the Transwell assay results. Data are presented as the mean ± SD, and the Student's t-test and one-way ANOVA were performed. *P<0.05, **P<0.01 and ***P<0.001. CtBP2, C-terminal-binding protein 2; LV, lentiviral; EdU, 5-ethynyl-2'-deoxyuridine.

Fig 5: Overexpression or knockdown of CtBP2 and TGIF in ECA109 cells. (A and D) ECA109 cells were observed under a fluorescence and phase contrast microscope following transfection. The recombinant lentiviral vectors contained the gene encoding the green fluorescent protein. Scale bar, 500 µm. (B and E) statistical analysis of percentage of positive cells in panels A and D. (C and F) mRNA expression levels of CtBP2 and TGIF in ECA109 cells following transfection was examined using RT-qPCR. (G and H) Expression levels of CtBP2 and TGIF in ECA109 cells following transfection were examined using western blot analysis. Data are presented as the mean ± SD, and the Student's t-test and one-way ANOVA were performed. *P<0.05, **P<0.01 and ***P<0.001. NC, negative control; CtBP2, C-terminal-binding protein 2; TGIF, transforming growth interacting factor; RT-qPCR, reverse transcription quantitative PCR; LV, overexpression of CtBP2 and TGIF; Sh, knockdown of CtBP2 and TGIF.