Fig 2: USP1 regulates the protein stability of C/EBPβ via deubiquitination.A, B Western blots of deubiquitination assays of C/EBPβ. AML12 cells were transfected with siRNA against Usp1 or treated with ML323 followed by 20 μM MG132 for 8 h. C Western blots of Ni2+ NTA pull-down show the ubiquitination of overexpressed C/EBPβ with 20 μM MG132 for 8 h in AML12 cells D Western blots of deubiquitination assays in AML12 cells after transfection with either the USP1 WT and C90S mutant followed by 20 μM MG132 for 8 h. E Western blot analysis of USP1 WT and C90S mutant dose-dependent effects on C/EBPβ in AML12 cells. Cells were transiently transfected with USP1 WT and C90S mutant F Western blots of deubiquitination assays in primary adipocytes isolated from the gWAT. SVF cells obtained from the gWAT were differentiated into adipocytes and treated with or without ML323. G The total polyubiquitinated proteins in HEK293 cells were captured by TUBE2 resins. Cells were treated with ML323 for 48 h followed by 20 μM MG132 for 8 h. Data represent the mean ± SEM (n = 3 for each lane) *P < 0.05 and **P < 0.01 for USP1 WT vs. USP1 C90S. Statistical significance was determined by two-tailed unpaired t test.

Fig 3: USP1 interacts with C/EBPβ and increases its protein stability.A, B Effects of Usp1 knockdown on the protein stability of C/EBPα and C/EBPβ. AML12 cells were transfected with siRNA against Usp1 or treated with ML323 for 48 h. Cells were then treated with or without 20 μM MG132 for 8 h. C Interaction between endogenous USP1 and C/EBPβ. The AML12 cells were immunoprecipitated using IgG and USP1 antibodies. D Immunoprecipitation of exogenous USP1 and C/EBPβ in HEK293 cells. Cell lysates were immunoprecipitated using IgG and USP1 antibodies and analyzed using western blotting. E Inhibition of interaction between endogenous USP1 and C/EBPβ by ML323. AML12 cells were treated with ML323 for 48 h and cell lysates were immunoprecipitated using IgG and USP1 antibodies. F HIS pull-down assay showing a direct interaction between USP1 and C/EBPβ. G, H Protein half-life of C/EBPβ by inhibition of USP1. AML12 cells were transfected with Usp1 siRNA or treated with ML323 for 48 h. Results were normalized using β-actin. Data represent the mean ± SEM (n = 3 for each lane) *P < 0.05, **P < 0.01, and *** P < 0.001 for scRNA vs. siUSP1. Statistical significance was determined by two-tailed unpaired t test.

Fig 4: Knockdown of Usp1 inhibits adipocyte differentiation.A Effect of Usp1 knockdown on adipocyte differentiation. The 3T3-L1 cells were transfected with siRNA for 48 h before DMI induction and differentiated for 6 days. Lipid accumulation was measured using ORO staining. Stained dye was eluted with 100% isopropanol and measured at OD500. B A change of cell numbers during mitotic clonal expansion by Usp1 knockdown in 3T3-L1 cells. Each sample was harvested 0, 24, and 48 h after DMI induction and cell numbers were counted using automatic cell counting machinery. C, D Quantitative RT-PCR and western blot analysis of adipogenic and lipogenic genes during adipocyte differentiation. E, F Protein and mRNA expression of USP1 and adipogenic factors by Usp1 overexpression. Mouse adipose-derived stromal vascular fraction cells from gWAT were transfected with the Usp1 plasmid. Results were normalized using β-actin. Data represent the mean ± SEM (n = 3 for each lane) *p < 0.05, **p < 0.01, and *** p < 0.001 for scRNA vs. siUsp1 and mock vs. Usp1. Statistical significance was determined by two-tailed unpaired t test.