Additional file 1 of E3 ubiquitin ligase RNF180 prevents excessive PCDH10 methylation to suppress the proliferation and metastasis of gastric cancer cells by promoting ubiquitination of DNMT1
Creators: name: Zhang, Nannan
name: Gao, Xiaoliang
name: Yuan, Qiangqiang
name: Fu, Xin
name: Wang, Pengliang
name: Cai, Fenglin
name: Liu, Hui
name: Zhang, Jing
name: Liang, Han
name: Nie, Yongzhan
name: Deng, Jingyu
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology

description: Additional file 1: Fig. S1. A PCDH10 protein levels in GC cell lines and GES-1. B The global methylation level of PCDH10 in gastric cancer cells from the Broad Institute CCLE databases. C The global methylation level of PCDH10 in gastric cancer tissues, adjacent non-tumor tissues from EWAS database. D The global methylation status of PCDH10 in gastric cancer tissues, adjacent non-tumor tissues confirmed by the NGS methylation analysis. E, F Western blot and qPCR confirmed that DNMT1 knockdown increased the protein and mRNA expression of PCDH10. Fig. S2. A PCDH10 overexpression led to an elevated expression of the cleaved form of caspase- 3 and poly (ADP-ribose) 2 polymerase (PARP) in HGC27 and AGS cells treated with staurosporine, which is an apoptosis-inducing reagent. B PCA for the expression profiles to distinguish the two groups (PCDH10 overexpression and control groups). C Volcano plot of altered gene expression patterns in PCDH10 overexpressed HGC27 cells identified from RNA-seq analysis. Fig. S3. A PCA for the expression profiles to distinguish the two groups (RNF180 overexpression and control groups. B Heatmap of 30 dysregulated genes identified from iTRAQ quantitative analysis. C The significant GO terms in the enrichment analysis of 30 dysregulated genes identified from iTRAQ quantitative analysis. D qPCRanalyses were used to detect the mRNA expression of DNMT1 in RNF180 overexpressed HGC27 cells and RNF180 silencing MKN45 cells. E qPCR analyses were used to detect the mRNA expression of PCDH10 in RNF180 overexpressed HGC27 cells. F Mutation in the RING domain of RNF180 did not affect the protein level of DNMT1 and PCDH10. G, H CCK8 and colonyforming assays confirmed that DNMT1 upregulation partially countervailed the inhibitive effect of RNF180 overexpression on the proliferation of HGC27 cells. I RNF180 lacking the RING domain failed to reverse MG132-induced increase in DNMT1 expression in HGC27 cells. Fig. S4. A Stable PCDH10 overexpressed HGC27 and AGS cells were established. B, C Stable RNF180 overexpressed HGC27 cells (B) and stable RNF180 knockdown MKN45 cells (C) were established. D, E, F Stable DNMT1 knockdown AGS cells (D), DNMT3A knockdown AGS cells (E), DNMT3B knockdown AGS cells (F) were established. G The expression of PCDH10 in gastric cancer cells from the Broad Institute CCLE databases. Fig. S5. A, B CCK-8 assay and colony formation was performed to determine the effect of RNF180 knockdown on cell proliferation.