Fig 2: USP43 regulates the migration and invasion of colorectal cancer cells. A-D. Cell scratch test, migration, and invasion assays analysis of USP43 knockdown treated SW480 cells and USP43 overexpression treated DLD1 cells. E-F. Western blot and qRT-PCR analysis of the EMT-related proteins (Ecad Ncad, and Vim in USP43 knockdown treated SW480 cells and USP43 overexpression treated DLD1 cells. **P<0.01, ***P<0.001.

Fig 3: IHC results of NAS-related genes (TMSB10 and VIM) and PD-L1. A Correlations between NAS and expression levels of TMSB10, VIM and PD-L1, respectively. B Representative IHC images of TMSB10 B, VIM C and PD-L1 D, respectively. Normal brain tissues (n = 3) and glioma tissues of WHO grade II (n = 3), III (n = 3) and IV (n = 3) were used. C Positive ratios of TMSB10, VIM and PD-L1 in IHC results, respectively. Bar = 50 µm

Fig 4: miR-574-5p may regulate the expression of VIM by direct binding. (A) miR-574-5p has two targeted binding areas with VIM according to bioinformatics prediction. (B) Immunofluorescence assay showed that miR-574-5p mimics and NC were successfully transfected into 293T cells (magnification, ×20); the transfected cells emitted green fluorescence. (B-a) 293T cell transfection with miR-574-5p NC for 48 h was observed under a light microscope. (B-b) 293T cells with miR-574-5p NC transfection for 48 h were observed under a fluorescence microscope. (B-c) 293T cells with miR-574-5p mimics transfection were observed under a light microscope at 48 h. (B-d) 293T cells with miR-574-5p mimics transfection were observed under a fluorescence microscope at 48 h. (C) Reverse transcription-quantitative PCR demonstrated that the miR-574-5p level was increased in 293T cells following transfection with miR-574-5p mimics (*P<0.05 vs. miRNA NC). (D) Dual luciferase reporter assay showed that miR-574-5p could regulate VIM expression by direct binding (**P<0.01 vs. miRNA NC). (E) miR-574-5p could reduce VIM expression at the protein level, according to the results of immunofluorescence analysis (magnification, ×200). VIM, vimentin; miR, microRNA; WT, wild-type; MUT, mutant; NC, negative control.

Fig 5: 5-Aza-CdR advances EMT in MCF7 cells via hypomethylation of Sipa1. (A) Effect of 5-Aza-CdR on the expression of SIPA1, E-cadherin and vimentin in MCF7 cells. The cells were treated with increasing concentrations of 5-Aza-CdR and the protein expression was determined by western blotting. (B) Effect of 5-Aza-CdR on the migration of MCF7 cells. The cells were treated with different concentrations of 5-Aza-CdR and the migrated cells were counted by the transwell assay. Each column represents the mean of triplicate experiments. **P<0.01, ***P<0.001; two-tailed unpaired Student's t-test. Error bars indicate s.d. Cells treated with DMSO were used as a negative control. (C-E) Effect of SIPA1 on the transcription of the genes including TGFB1 (TGFß), CTNNB1 (ß-catenin), SNAI1, SNAI2, ZEB1, CDH1, CDH2 and VIM in breast cancer cells. The mRNA level was determined through qRT-PCR in parent and SIPA1 knockdown MDA-MB-231 cells (C), parent and SIPA1 knockdown BT-549 cells (D), and parent and SIPA1-overexpressed MCF7 cells (E). Each column represents the mean of five separate experiments. Error bars indicate s.d. *P<0.05, **P<0.01, ***P<0.001; ns, no significance; two-tailed unpaired Student's t-test.