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.

Fig 5: Regulation of DNA damage signaling and FANCD2 recruitment by BAP1 and USP1.A Immunofluorescence detection of γ-H2AX (green) with Alexa647 conjugated antibody in H28-Venus and H28-BAP1 cells after 72 h infected with each lentivirus. Nuclei were stained in blue with 4’,6-diamidino-2-phenylindole (DAPI). The γ-H2AX positive cells were counted with over 5 foci. γ-H2AX positive cell ratio was normalized total number of DAPI. Values are expressed as means ± SD. Statistical significance compared with H28-Venus and H28-BAP1 with non-target or shUSP1 was determined using Tukey-Kramer test: **P < 0.01. Scale bars indicated 20 μm. B Immunofluorescence detection of FANCD2 (red) with Alexa546 conjugated antibody. Nuclei were stained in blue with DAPI. The FANCD2 positive cells were counted as nuclei with red foci. FANCD2 positive cell ratio was normalized total number of DAPI. Data are presented as the means ± SD. Statistical significance compared with H28-Venus and H28-BAP1 at non-target or shUSP1 was determined using Tukey-Kramer test: **P < 0.01. Scale bars indicated 20 μm. C The ratio of co-localization of γ-H2AX foci and FANCD2 foci upon USP1 knockdown in H28 and H28-BAP1 cells. The ratio of FANCD2 foci colocalized with γ-H2AX was normalized the total number of γ-H2AX. Values are expressed as means ± SD. Scale bars indicated 20 μm.