Cdh1 KO-activated Ezh2 promotes gastric tumorigenesis. (A) Integrated batch-based and regulon pattern-based UMAP for WT, KP, and EKP GOs. Six transcriptional modules were identified. (B) Separated regulon patterns based UMAP for WT, KP, and EKP GOs. (C) Flow chart of regulons selection process. (D) Regulons enriched in WT, KP, and EKP GOs, based on Z Score. 32 regulons were highly expressed in EKP samples compared to WT and KP. (E) Regulons enriched in WT, KP, and EKP GOs, based on RSS. The top 20 were selected by Z score. The whole regulon list based on RSS is shown in Table S18. (F) Venn diagram for the regulons from D and E. 20 regulons were overlapped. (G) Dot plot of the regulons (WT, KP, and EKP GOs) increased in TCGA DGAC patients. (H) Regulon activity-based UMAP of Ezh2 in WT, KP, and EKP GOs. The cells with lighter color represent regulated by Ezh2. (I and J) Dot plots of Ezh2 downstream target genes (I, genes which are downregulated by EZH2 activation through histone modification; J, genes which are downregulated by EZH2 activation reported in gastric cancer) scores in the epithelial cells of DGAC1 and DGAC2. P values were calculated by using a Mann–Whitney testing. Gene list of EZH2 targeted genes was listed in Table S9. (K) The level of H3K27Ac and H3K27Me3 expression in KP and EKP allografts. Quantification was displayed. Scale bars: 20 μm. (L) Crystal violet staining of KP and EKP cells after GSK343 (EZH2 inhibitor, 10 μM, 96 h). (M) Bright-field images of KP and EKP GOs after treatment with GSK343 (EZH2 inhibitor, 10 μM, 96 h). D2: day 2; D6: day 6. Scale bars: 200 μm. (N) Statistical analysis of KP and EKP gastric organoid size and number in response to GSK343 treatment. The number of organoids (right Y-axis) and their size (left Y-axis) were assessed following treatment with GSK343. On day 2 (D2), the number of organoids was determined for the image depicted in M, and this count was considered as 100% (n numbers are presented in the bubble plot). On day 6 (D6), the number of organoids in the same field for each group was counted (n numbers also displayed in the bubble plot). The percentage of each group on D6 was calculated by dividing the number of viable organoids at D6 by the number at D2. The viable percentage is presented in the bar graph. (O–Q) Transplantation of EKP cells followed by EZH2 inhibition. (O) Bright-field images of EKP allograft tumors treated with DMSO and GSK343 (20 mg/kg) separately (n = 3). (P) Tumor growth curve of EKP allografts treated with DMSO and GSK343 (20 mg/kg) after cell subcutaneous transplantation. (Q) Tumor mass of EKP allografts treated with DMSO and GSK343 (20 mg/kg) after mice scarification. P values were calculated using Student’s t test; error bars: SD. ns: P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001. All data are derived from two or more independent experiments with the indicated number of samples.
Figure 5.

Cdh1 KO-activated Ezh2 promotes gastric tumorigenesis. (A) Integrated batch-based and regulon pattern-based UMAP for WT, KP, and EKP GOs. Six transcriptional modules were identified. (B) Separated regulon patterns based UMAP for WT, KP, and EKP GOs. (C) Flow chart of regulons selection process. (D) Regulons enriched in WT, KP, and EKP GOs, based on Z Score. 32 regulons were highly expressed in EKP samples compared to WT and KP. (E) Regulons enriched in WT, KP, and EKP GOs, based on RSS. The top 20 were selected by Z score. The whole regulon list based on RSS is shown in Table S18. (F) Venn diagram for the regulons from D and E. 20 regulons were overlapped. (G) Dot plot of the regulons (WT, KP, and EKP GOs) increased in TCGA DGAC patients. (H) Regulon activity-based UMAP of Ezh2 in WT, KP, and EKP GOs. The cells with lighter color represent regulated by Ezh2. (I and J) Dot plots of Ezh2 downstream target genes (I, genes which are downregulated by EZH2 activation through histone modification; J, genes which are downregulated by EZH2 activation reported in gastric cancer) scores in the epithelial cells of DGAC1 and DGAC2. P values were calculated by using a Mann–Whitney testing. Gene list of EZH2 targeted genes was listed in Table S9. (K) The level of H3K27Ac and H3K27Me3 expression in KP and EKP allografts. Quantification was displayed. Scale bars: 20 μm. (L) Crystal violet staining of KP and EKP cells after GSK343 (EZH2 inhibitor, 10 μM, 96 h). (M) Bright-field images of KP and EKP GOs after treatment with GSK343 (EZH2 inhibitor, 10 μM, 96 h). D2: day 2; D6: day 6. Scale bars: 200 μm. (N) Statistical analysis of KP and EKP gastric organoid size and number in response to GSK343 treatment. The number of organoids (right Y-axis) and their size (left Y-axis) were assessed following treatment with GSK343. On day 2 (D2), the number of organoids was determined for the image depicted in M, and this count was considered as 100% (n numbers are presented in the bubble plot). On day 6 (D6), the number of organoids in the same field for each group was counted (n numbers also displayed in the bubble plot). The percentage of each group on D6 was calculated by dividing the number of viable organoids at D6 by the number at D2. The viable percentage is presented in the bar graph. (O–Q) Transplantation of EKP cells followed by EZH2 inhibition. (O) Bright-field images of EKP allograft tumors treated with DMSO and GSK343 (20 mg/kg) separately (n = 3). (P) Tumor growth curve of EKP allografts treated with DMSO and GSK343 (20 mg/kg) after cell subcutaneous transplantation. (Q) Tumor mass of EKP allografts treated with DMSO and GSK343 (20 mg/kg) after mice scarification. P values were calculated using Student’s t test; error bars: SD. ns: P > 0.05; *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001. All data are derived from two or more independent experiments with the indicated number of samples.

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