Journal: Cells

Article Title: Ubiquitin-Specific Protease 1 Promotes Bladder Cancer Progression by Stabilizing c-MYC

doi: 10.3390/cells13211798

Figure Lengend Snippet: USP1 overexpression promotes bladder cancer cell proliferation, migration, and invasion. ( A ) T24 cells were transfected with FLAG-USP1 or FLAG-USP1 C90S, as indicated, and protein levels were measured by Western blotting. GAPDH served as a control. ( B ) CCK-8 assays were used to analyze cell proliferation ( n = 6). ( C ) Colony formation assays were performed to evaluate cell viability. The colonies were stained with crystal violet and photographed. The number of colonies was determined and plotted ( n = 3). ( D ) Transwell experiments were used to evaluate the effects of USP1 overexpression on cell migration. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). The data (mean ± SEM) are representative of 3 independent experiments. ( E ) Transwell chambers with Matrigel were used to evaluate the effects of USP1 overexpression on cell invasion. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). The data (mean ± SEM) are representative of 3 independent experiments. Statistical significance was analyzed by ANOVA or Student’s t test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody (pAb) against USP1 (14346-1-AP) and mouse mAb against GAPDH (60004-1) were purchased from Proteintech (Wuhan, China).

Techniques: Over Expression, Migration, Transfection, Western Blot, Control, CCK-8 Assay, Staining

Journal: Cells

Article Title: Ubiquitin-Specific Protease 1 Promotes Bladder Cancer Progression by Stabilizing c-MYC

doi: 10.3390/cells13211798

Figure Lengend Snippet: USP1 deficiency inhibits cell proliferation, migration, and invasion. ( A ) USP1 protein levels in WT and USP1-deficient UMUC3 cells were measured by Western blotting, with GAPDH as a loading control. ( B ) Colony formation assays showed the viability of USP1-deficient UMUC3 bladder cancer cells. Colonies were stained with crystal violet and subsequently imaged (left). The number of colonies was determined and plotted (right, n = 3). ( C ) Cell proliferation was analyzed by CCK-8 assays with daily measurements for 5 days ( n = 6). ( D ) Transwell experiments were used to evaluate the effects of USP1 deficiency on UMUC3 bladder cancer cell migration. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). ( E ) Transwell chambers with Matrigel were used to evaluate the effects of USP1 deficiency on UMUC3 bladder cancer cell invasion. The cells were imaged (left, ×20 magnification) and counted, and the results were plotted (right, n = 3). The data (mean ± SD) are representative of 3 independent experiments. Statistical significance was analyzed by ANOVA or Student’s t test. *** p < 0.001.

Article Snippet: Rabbit polyclonal antibody (pAb) against USP1 (14346-1-AP) and mouse mAb against GAPDH (60004-1) were purchased from Proteintech (Wuhan, China).

Techniques: Migration, Western Blot, Control, Staining, CCK-8 Assay

Journal: Cells

Article Title: Ubiquitin-Specific Protease 1 Promotes Bladder Cancer Progression by Stabilizing c-MYC

doi: 10.3390/cells13211798

Figure Lengend Snippet: USP1 interacts with c-MYC and promotes c-MYC protein stability. ( A ) Immunofluorescence images of HA-USP1 (red) and FLAG-c-MYC (green) in HEK293T cells. DAPI was used as a nuclear stain (blue). ( B , C ) Immunoprecipitation experiments showed the interaction between USP1 and c-MYC in HEK293 cells. ( D ) Western blot analysis of c-MYC expression in USP1-overexpression T24 cells. ( E ) Western blot analysis of c-MYC expression in USP1-deficient UMUC3 cells. ( F ) Cells were transfected with increasing amounts of the HA-USP1 (0, 200, 400, and 800 ng), HA-USP1 C90S (400 and 800 ng), and FLAG-c-MYC plasmids, and Western blotting was performed to determine the effect of USP1 protein levels on c-MYC expression in HEK293T cells. ( G ) Cells were transfected with FLAG-c-MYC with HA-USP1 or HA-USP1 C90S, as indicated. Western blot analysis of c-MYC stability after treatment with CHX (50 μg/mL) for the indicated time. GAPDH served as a control. ( H ) Cells were co-transfected with FLAG-c-MYC and Myc-Ub with or without HA-USP1 or HA-USP1 C90S, treated with MG132 (10 μM) for 6 h, and then subjected to ubiquitination assays.

Article Snippet: Rabbit polyclonal antibody (pAb) against USP1 (14346-1-AP) and mouse mAb against GAPDH (60004-1) were purchased from Proteintech (Wuhan, China).

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Expressing, Over Expression, Transfection, Control, Ubiquitin Proteomics