Fig 2: Analysis of ECAD, SPINK1, and their combination to predict CSPH.(A) ECAD and (B) SPINK1 in plasma of patients with advanced chronic liver disease with subclinical portal hypertension (HVPG <10; n = 11) or with CSPH (HVPG ≥10; n = 36) of three different aetiologies: MASH, OH, or HCV. (C) ROC curve of ECAD, SPINK1, and ES and (D) its performance. Data were compared using Student’s t test (∗p <0.05). Values of AUROC, the 95% CI, the cut-off value with better Sens and Spec, PPV, and NPV. AUROC, area under the receiver operating characteristics; CSPH, clinically significant portal hypertension; ECAD, E-cadherin; ES, ECAD + SPINK1; HVPG, hepatic venous pressure gradient; MASH, metabolic-associated steatohepatitis; NPV, negative predictive value; OH, alcohol-associated; PPV, positive predictive value; ROC, receiver operating characteristic; Sens, sensitivity; Spec, specificity; SPINK, serine protease inhibitor Kazal-type 1.

Fig 3: Effects of PD98059 on E-cadherin and spheroidogeneis of WT and CDH1 KO HCT116 cells. (A) PD98059 decreased cell viability in dose-dependent manner with a significant difference between WT and CDH1 KO HCT116 cells while ERK activation was similar. (B) PD98059 (20 µM) inhibited ERK activation in both cells, but increased E-cadherin expression. (C) PD98059 decreased sizes of spheroids in both cell lines. (D) PD98059 significantly increased the expression of β-catenin and pan-RAS without affecting the expression of EGFR. Scale bar=200 µm. *P<0.05, **P<0.01, ***P<0.001 vs. vehicle; #P<0.05, ###P<0.001 vs. WT. WT, wild-type; KO, knock-out.

Fig 4: Long-term spheroidogenesis of WT and CDH1 KO HCT116 cells. (A) When spheroidogenesis was maintained for 21 days, the number of spheroids was decreased while sizes of spheroids were increased in a time-dependent manner in both cells. Interestingly, some spheroids from CDH1 KO cell line lost their morphological integrity (arrows). Scale bar=500 µm. (B) Immunocytochemistry (magnification, ×4 and ×20) using with an anti-E-cadherin antibody for spheroids revealing decreased E-cadherin in CDH1 KO cells. Arrows indicate necrosis. *P<0.05, ***P<0.001 vs. Day 7. WT, wild-type; KO, knock-out.

Fig 5: General characteristics of WT and CDH1 KO HCT116 cells. (A) CDH1 KO was confirmed by western blotting. (B) Proliferation was similar between two cell types. (C) Respective cell viability after treatment with an anticancer drug, 5-FU, epidermal growth factor receptor inhibitor (erlotinib) or src kinase inhibitor (saracatinib) was similar between two cell lines. ***P<0.001 vs. WT; #P<0.05, ##P<0.01, ###P<0.001 vs. vehicle. WT, wild-type; KO, knock-out; 5-FU, 5-fluorouracil.