Fig 1: Schematic mechanism illustrating USP14-promoted NAFLD.USP14 stabilises HSP90AA1 by deubiquitination. stabilised HSP90AA1 promotes mitochondrial dysfunction and lipid peroxidation by increasing CYP2E1 proteins in NAFLD . Ub, ubiquitination modification.
Fig 2: USP14 reduces K48-linked ubiquitination of HSP90AA1 to stabilize it.A USP14 and CYP2E1 protein expression in AML12 cells treated with MG132 and CQ and transfected with the corresponding vectors. B Co-IP assay to assess ubiquitination of HSP90AA1 in HEK-293T cells transfected with the corresponding labeled USP14, HSP90AA1, or ub plasmids. C Construction of an active site mutant of USP14 (USP14 C114A) and a truncation mutant of USP14 lacking the UBL. D Co-IP analysis of the interaction domain of USP14 and HAP90AA1. E Co-IP analysis of the interaction domain of USP14 on HAP90AA1 deubiquitination. F Identification of the type of HAP90AA1 deubiquitination by USP14. G Analysis of protein levels of CYP2E1 after AML12 cells were transfected with Ad-CMV, Flag-USP14 FL, Flag-C114A, and Flag-?UBL. **P < 0.01 between the two groups. NS not significant.
Fig 3: USP14 increases CYP2E1 by promoting HSP90AA1 protein levels.A, B Exogenous co-IP assays were performed to assess the interaction between USP14 or HSP90AA1 in HEK-293T cells transfected with the corresponding labeled USP14 or HSP90AA1 plasmids. C Representative endogenous co-IP analyses show the binding of USP14 to HSP90AA1 in PO-treated AML12 cells for 12 h. IgG was used as the control. D Immunofluorescence analysis showing co-localization of USP14 and HSP90AA1 in HEK-293T cells transfected with Flag-USP14 and MYC-HSP90AA1, scale bar, 50 µm. E PO treatment of protein levels of USP14 and HSP90AA1 in AML12 cells. F HAP90AA1 protein levels in hepatocytes after USP14 overexpression and knockdown. G Protein levels of HSP90AA1 in AML12 cells treated with 100 µg/mL cycloheximide for the corresponding time and transfected with Flag-USP14 plasmids or empty vectors. H Exogenous co-IP assays were performed to assess the interaction between HSP90AA1 or CYP2E1 in HEK-293T cells transfected with the corresponding labeled HSP90AA1 or CYP2E1 plasmids. I Protein levels of CYP2E1 in AML12 cells treated with 100 µg/mL cycloheximide for the corresponding time and transfected with Flag-HSP90AA1 plasmids or empty vectors. J Effect of AML12 cells cotransfected with Flag-USP14 and HSP90AA1 siRNA on CYP2E1 protein levels. *P < 0.05 between the two groups. **P < 0.01 between the two groups.
Fig 4: Interaction between CERS1 and transcription factor USP14. (A) Expression of indicated proteins was evaluated using western blotting. (A) USP14 expression following CERS1 knockdown was detected using western blotting. (B) Co-IP demonstrated an interaction between CERS1 and USP14. (C) Immunofluorescence was performed to detect CERS1 and USP14 localization in cells.
Fig 5: USP14 induces LPO and inflammation by promoting CYP2E1 protein levels.A AML12 cells were PO-treated and transfected with the corresponding adenovirus for BODIPY staining (n = 3), scale bar, 200 µm. B–D ATP, NAD+/NADH, and MDA levels in AML12 cells treated with PO and transfected with the corresponding adenovirus. E Quantitative PCR analysis of the expression of inflammatory genes in each group of cells. F Assessment of ROS and mitochondrial ROS in AML12 cells (n = 3), ROS scale bar, 50 µm; other scale bar, 100 µm. G, H Liver MDA and SOD levels in mice after HFD induction and injection of the corresponding adenovirus. I Schematic representation of the function of CYP2E1. J protein expression of CYP2E1 from the liver of each group of mice. K, L USP14 and CYP2E1 protein expression in AML12 cells after being treated with PO and transfected with the corresponding vectors. M CYP2E1 protein levels of AML12 cells treated with 100 µg/mL cycloheximide for the corresponding time and transfected with Flag-USP14 plasmids or empty vectors. **P < 0.01 for the vector group compared to the Flag-USP14 group. NS not significant.
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