p5091  (MedChemExpress)

Journal: Cell Death & Disease

Article Title: USP7 depletion potentiates HIF2α degradation and inhibits clear cell renal cell carcinoma progression

doi: 10.1038/s41419-024-07136-0

Figure Lengend Snippet: a ccRCC cells were seeded in 96-well plates, 12 h later, the cells were treated with the indicated concentration of P5091 or P22077 for 24 h, and cell viability was measured using CCK-8. b ccRCC cells with or without USP7 knockdown were seeded in 96-well plates, and cell proliferation was measured using CCK-8. c and d OS-RC-2 cells with or without USP7 knockdown were resuspended in 200 μL PBS containing 50% matrigel and subcutaneously injected into nude mice ( n = 6 for each group), the tumor growth ( c ) and tumor weight ( d ) were analyzed. e and f OS-RC-2 xenograft tumor with or without treatment of P5091 (i.p. 25 mg/kg), tumor growth curve and tumor images ( e ), and tumor weight ( f ) from mice subjected to indicated treatments were shown ( n = 6 for each group). g Diagram of the process of establishing VHL mutant ccRCC PDO and mini-PDX model from patient tumors for in vitro and in vivo drug treatment. h ccRCC PDOs were seeded in 96 well plates, 48 h later, PDOs were treated with the indicated concentration of P5091 or P22077 for 72 h, Representative PDO images were shown (left) and the cell viability was analyzed using CellTiter-Glo® Luminescent Cell Viability Assay (right). Scale bar, 50 μm. i Primary ccRCC cells were loaded into capsules and implanted into nude mice for constructing the mini-PDX model, mice were administered with saline or P5091(i.p. 25 mg/kg) for 7 days, relative tumor viability was detected using the CellTiter-Glo® Luminescent Cell Viability Assay. The experiments were independently repeated three times with similar results ( a , b , and h ). Data are shown in mean ± SD, ** p < 0.01.

Article Snippet: Cycloheximide (HY-12320), P5091 (HY-15667), and P22077 (HY-13865) were obtained from MedChemExpress.

Techniques: Concentration Assay, CCK-8 Assay, Knockdown, Injection, Mutagenesis, In Vitro, In Vivo, Cell Viability Assay, Capsules, Saline

Journal: Cell Death & Disease

Article Title: USP7 depletion potentiates HIF2α degradation and inhibits clear cell renal cell carcinoma progression

doi: 10.1038/s41419-024-07136-0

Figure Lengend Snippet: a ccRCC cell lines were treated with the indicated concentration of P5091 or P22077 for 12 h, and the endogenous HIF2α protein level was analyzed by immunoblot using ACTIN as the loading control. b ccRCC cell lines were infected with control shRNA or USP7 shRNAs, and the endogenous USP7 and HIF2α protein levels were analyzed by immunoblot using ACTIN as the loading control. c 293T cells were transfected with indicated plasmids for 24 h and treated with cycloheximide (CHX, 100 μg/ml) for the indicated time, Flag-HIF2α level was analyzed and quantified by immunoblot with ACTIN as a loading control. d OS-RC-2 cells with or without USP7 knockdown were treated with CHX (100 μg/ml) for the indicated time, and endogeneous HIF2α level was analyzed and quantified by immunoblot with ACTIN as a loading control. e Immunoblots showing USP7, HIF2α, VEGFA, and CCND1 in xenograft tumors with or without USP7 knockdown by shRNA, six tumors were used per group, and ACTIN was used as a loading control. f IHC analysis of USP7 and HIF2α expression in a tissue microarray containing 36 ccRCC samples, representative images were shown (left) and the correlation between USP7 and HIF2α was analyzed (right). Scale bar, 100 μm. g Cells were infected with shRNAs targeting USP7 and/or HIF2α, and cell proliferation was assessed using CCK8. h Tube formation ability of HUVEC cultivated for 4 h in the conditional medium from infected OS-RC-2 cells, cells were treated with Calcein- AM. Scale bar, 50 μm. Pictures were taken with a fluorescence microscope, and tube numbers were counted. The experiments were independently repeated three times with similar results ( a – d , g and h ). Data are shown in mean ± SD. ** p < 0.01, NS means no significant difference.

Article Snippet: Cycloheximide (HY-12320), P5091 (HY-15667), and P22077 (HY-13865) were obtained from MedChemExpress.

Techniques: Concentration Assay, Western Blot, Control, Infection, shRNA, Transfection, Knockdown, Expressing, Microarray, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: USP7 depletion potentiates HIF2α degradation and inhibits clear cell renal cell carcinoma progression

doi: 10.1038/s41419-024-07136-0

Figure Lengend Snippet: a List of drugs used for combined drug screening. b Cells were seeded in 96-well plates with 1*10 4 cells per well, 24 h later, cells were subjected to indicated concentration of afatinib with or without the combination of P5091 (10 μM) for 24 h, and cell viability was analyzed using CCK-8. c Cells with or without USP7 depletion were seeded in 96-well plates with 1*10 4 cells per well, 24 h later, cells were treated with the indicated concentration of afatinib for another 24 h and then cell viability was measured using CCK-8. d ccRCC cells were treated with USP7 inhibitor and/or Afatinib for 48 h before cell death analysis (right) by Annexin V–PI staining. e Combination Index (CI) of P5091/P22077 and Afatinib was analyzed in ccRCC cells using the CompuSyn software (Biosoft). f ccRCC PDOs were seeded in 96-well plates, 48 h later, the PDOs were subjected to the single or combinational treatment of P22077 (1 μM), P5091 (1 μM), and Afatinib (1 μM) for 10 days, representative images of PDOs were shown. Scale bar, 75 μm. g PDO cell viability in ( f ) was measured by CellTiter-Glo® Luminescent Cell Viability Assay. The experiments were independently repeated three times with similar results and the graph shows mean ± SD from triplicates ( b – d and f ). ** p < 0.01.

Article Snippet: Cycloheximide (HY-12320), P5091 (HY-15667), and P22077 (HY-13865) were obtained from MedChemExpress.

Techniques: Concentration Assay, CCK-8 Assay, Staining, Software, Cell Viability Assay

Journal: Cell Death & Disease

Article Title: USP7 depletion potentiates HIF2α degradation and inhibits clear cell renal cell carcinoma progression

doi: 10.1038/s41419-024-07136-0

Figure Lengend Snippet: a Cells were treated with the indicated concentration of Afatinib for 24 h, and HIF2α protein levels were analyzed by immunoblots with ACTIN as loading control. b Cells with or without the pretreatment of 5 μM Afatinib for 24 h were subjected to the administration of CHX (100 μg/ml) for the indicated time, and HIF2α levels were analyzed and quantified with ACTIN as control. c Cells were pretreated with the indicated concentration of Afatinib for 24 h, and then treated with 20 μM MG132 for 6 h, the polyubiquitination level of HIF2α was analyzed by denaturing immunoprecipitation and immunoblots. d and e Cells were treated with the indicated concentration of Afatinib for 24 h, and FUBP1, FUBP3, and USP7 expression levels were analyzed by qRT-PCR ( d ) and immunoblots ( e ). f 786-O cells with or without Flag-USP7 expression were treated with 10 μM Afatinib for 24 h, and protein expression was analyzed using immunoblots. g 786-O cells with or without USP7 expression were pretreated with 5 μM Afatinib for 24 h, and then subjected to the administration of CHX (100 μg/ml) for the indicated time, HIF2α levels were analyzed and quantified with ACTIN as control. h Single or combinational treatment of P5091 (5 μM), P22077 (5 μM), and Afatinib (5 μM) for 24 h, HIF2α protein levels were analyzed by immunoblots with ACTIN as loading control. i Proposed a mechanism for the action of FUBP1/3-USP7-HIF2α regulatory axis in ccRCC tumor progression and the mechanism-based targeted strategy. The experiments were independently repeated three times with similar results ( a – h ), and the Graph shows mean ± SD from triplicates in one experiment ( d ). ** p < 0.01, * p < 0.05.

Article Snippet: Cycloheximide (HY-12320), P5091 (HY-15667), and P22077 (HY-13865) were obtained from MedChemExpress.

Techniques: Concentration Assay, Western Blot, Control, Immunoprecipitation, Expressing, Quantitative RT-PCR