Fig 1: ZNF582 overexpression inhibits ccRCC cell growth and metastasis by regulating TJP2 in vivo.A Representative bioluminescence imaging of ZNF582 overexpression and control groups and quantification of these bioluminescence imaging. B Quantification of the tumor weight of ZNF582 overexpression and control groups. C Representative picture of HE (100X, Bar: 100 um), EdU and TUNEL expression (400X, Bar: 25 um) in the kidney tumors of ZNF582 overexpression and control groups and quantification the expression of EdU and TUNEL. D Representative lung ex vivo bioluminescence imaging of ZNF582 overexpression and control groups and quantification of ex vivo imaging. E Representative picture of HE (100X, Bar: 100 um), E-cadherin and N-cadherin expression (400X, Bar: 25 um) in the lung metastases of ZNF582 overexpression and control groups and quantification of these picture. IOD: integrated optical density. F Representative bioluminescence imaging of TJP2 knockdown and control groups and quantification of these bioluminescence imaging. G Quantification of the tumor weight of TJP2 knockdown and control groups. H Representative picture of HE (100X, Bar: 100 um), EdU and TUNEL expression (400X, Bar: 25 um) in the kidney tumors of TJP2 knockdown and control groups and quantification the expression of EdU and TUNEL. I Representative lung ex vivo bioluminescence imaging of TJP2 knockdown and control groups and quantification of ex vivo imaging. J Representative picture of HE (100X, Bar: 100 um), E-cadherin and N-cadherin expression (400X, Bar: 25 um) in the lung metastases of TJP2 knockdown and control groups and quantification of these picture. IOD: integrated optical density. K Schematic model of ZNF582 overexpression up-regulates TJP2 protein expression, which leads to up-regulation of ERK2 protein and down-regulation of p-ERK2, therefore suppressing the growth and metastasis of ccRCC. All result data are represented as mean ± SEM.
Fig 2: Combination of TJP2 and ERK2 inhibits ERK2 phosphorylation.A Detection of p-ERK2 protein expression in ZNF582 overexpression and control OSRC2 and Caki-1 cells. B Detection of p-ERK2 protein expression in TJP2 knockdown and control OSRC2 and Caki-1 cells. C, D Detection of MEK1/2 and p-MEK1/2 protein expression in TJP2 overexpression and control OSRC2 and Caki-1 cells. E Detection of the expression of ERK2 binding to MEK1/2 in TJP2 overexpression and control OSRC2 and Caki-1 cells. F, G Comparison of the expression of BCL-2, Cleaved Caspase-3, N-cadherin and E-cadherin between ZNF582 overexpression and control OSRC2 and Caki-1 cells. H, I Comparison of the expression of BCL-2, Cleaved Caspase-3, N-cadherin and E-cadherin between TJP2 knockdown and control OSRC2 and Caki-1 cells.
Fig 3: ZNF582 protein binds to TJP2 and ERK2 protein and regulates their expression.A Comparison of the differential protein between ZNF582 overexpression and control OSRC2 cells by TMT experiment. B Identification of the possible binding proteins of ZNF582 protein by Co-IP, silver-staining and mass spectrometry. C Venn diagram analysis of TMT results and mass spectrometry results. D TJP2 expression is significantly positively correlated with ZNF582 expression based on GSE126964, GSE53757 and TCGA-KIRC data, and MAPK1 (ERK2) expression is significantly positively correlated with TJP2 expression based on TCGA-KIRC data. E, F Confirmation of ZNF582 protein binds to TJP2 and ERK2 protein in OSRC2 and Caki-1 cells by IP and Western Blot. G, H Comparison of TJP2 and ERK2 expression between ZNF582 overexpression and control OSRC2 and Caki-1 cells.
Fig 4: Knockdown of TJP2 expression reverses the phenotype inhibition of ccRCC cell caused by ZNF582 overexpression in vitro.A Comparison of the number of cell clones in TJP2 knockdown and control OSRC2 and Caki-1 cells. Comparison of the proportion of EdU (B) and TUNEL (C) positive cells in TJP2 knockdown and control OSRC2 and Caki-1 cells by immunofluorescence (200X, Bar: 50 um). D Wound healing assay determined the migratory distances of TJP2 knockdown and control OSRC2 and Caki-1 cells (100X, Bar: 100 um). Comparison of cell migration (E) and cell invasion (F) ability in TJP2 knockdown and control OSRC2 and Caki-1 cells (100X, Bar: 100 um). The presentation of the histogram was the result of three independent experiments. **P < 0.01, ***P < 0.001, compared with the corresponding control.
Fig 5: ZNF582 overexpression inhibits the growth and migration of ccRCC cell in vitro.A Comparison of the number of cell clones in ZNF582 overexpression and control OSRC2 and Caki-1 cells. Comparison of the proportion of EdU (B) and TUNEL (C) positive cells in ZNF582 overexpression and control OSRC2 and Caki-1 cells by immunofluorescence (200X, Bar: 50 um). D Wound healing assay determined the migratory distances of ZNF582 overexpression and control OSRC2 and Caki-1 cells (100X, Bar: 100 um). Comparison of cell migration (E) and cell invasion (F) ability in ZNF582 overexpression and control OSRC2 and Caki-1 cells (100X, Bar: 100 um). The presentation of the histogram was the result of three independent experiments. **P < 0.01, ***P < 0.001, **** P < 0.0001, compared with the corresponding control.
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