Fig 1: USP14 interacts with MDM2 and upregulates MDM2 protein level. (A) Immunofluorescent staining of GFP was executed after co-transfected with cherry-MDM2 and flag-USP14 plasmids for 24 h in HeLa cells. The co-localization between USP14 and MDM2 was revealed by immunofluorescent staining. (B) Co-immunoprecipitation (Co-IP) assay for the interaction between USP14 and MDM2 in HeLa cells. Total proteins were extracted from HeLa cells, immunoprecipitated with USP14 antibody beads and immunoblotted for MDM2. (C-F) After transfected with pLVX-puro vector or pLVX-USP14-HA for 48 h, the levels of HA (USP14) and MDM2 protein were detected by western blot in HeLa cells. (G-J) The HeLa or 293T cells were transfected with pLKO.1-shUSP14 or pLKO.1-EGFP-Puro vector (scramble shRNA control) for 48 h, and then the levels of USP14 and MDM2 protein were measured by western blot. *p < 0.05; **p< 0.01.
Fig 2: circARHGAP10 act as a scaffold for the interaction between TRIM25 and MAT2A to facilitate ubiquitination of MAT2A.A Intersection of the proteomics between T24 and T24-CR cells and the mass spectrometry analysis of circARHGAP10 RNA-pulldown. B Differential protein levels of ISG15, USP5, USP14 and TRIM25 of T24 and T24-CR cells identified with proteomics analysis. C Binding of circARHGAP10 with TRIM25 in BCa cells. D Co-localization of TRIM25 with MAT2A in BCa cells. Scale bar = 10 µm. E Co-IP validated TRIM25 binding with MAT2A in T24-CR cells. F Molecular docking of the protein structure of TRIM25 and MAT2A. G MAT2A protein expression in BCa cells with knockdown of TRIM25. H Protein levels of MAT2A in BCa cells with the over-expression of TRIM25 or TRIM25?RBD. I Co-IP experiment was performed to identify the ubiquitination modification level of MAT2A in BCa cells with the over-expression of TRIM25 or TRIM25?RBD. J Co-IP experiment was performed to identify the ubiquitination level of MAT2A in 293 T cells with the over-expression of TRIM25 or TRIM25?RBD. K Co-IP experiment was performed to identify the ubiquitination level of MAT2A in 293 T cells with the over-expression of HA-Ubiquitin or the indicated mutants. L Co-IP experiment was performed to identify the ubiquitination level of MAT2A in BCa cells with the over-expression of TRIM25 or the silencing of circARHGAP10. M The protein level of MAT2A in BCa cells with the over-expression of TRIM25 or the silencing of circARHGAP10. N Co-IP experiment was performed to validate the correlation of circARHGAP10 with binding of TRIM25 with MAT2A in T24-CR cells. RNase A (10 µg/ml) and RNase R (100 U/ml) were the indicated treatment concentration.
Fig 3: A mechanism scheme of USP14 in endometrial carcinoma.
Fig 4: miR-124-3p targeted and repressed USP14. (A) StarBase was employed to analyze the upstream miRNAs of USP14, and a Venn diagram was adopted to analyze the USP14-targeted miRNAs shared by miRmap, microT, miRanda, PicTar, and TargetScan. (B) The binding sites between miR-124-3p and USP14. (C) The targeting correlation between miR-124-3p and USP14 was verified by dual-luciferase reporter assay. (D, E) miR-124-3p mimics were transfected into EC cells, and USP14 mRNA and protein expression was assessed. NS, **, ***represent P > 0.05, P < 0.01, and P < 0.001. N = 3.
Fig 5: The effects of USP14 knockdown in the apoptosis, migration and invasion of EC cells. (A) sh-USP14 or sh-NC was transfected into HUC-1-B and SNG-M cells, and the USP14 profile in EC cell lines was examined by WB. (B) Colony formation of HEC-1-B and SNG-M cells was tracked by colony formation experiments. (C) FCM was used to observe apoptosis. (D) The expression levels of Bcl-2, Bax, and Cyclin D1 were examined by WB. (E) Transwell assays were implemented to evaluate EC cell migration and invasion. (F) E-cadherin, Vimentin, Snail1, ZEB1, and Slug profiles were examined by WB. NS, *, **, ***indicate P > 0.05, P < 0.05, P < 0.01, and P < 0.001, respectively, vs. sh-NC. N = 3.
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