osrc2 cell line  (Procell Inc)

Journal: Neoplasia (New York, N.Y.)

Article Title: CYP1B1 promotes angiogenesis and sunitinib resistance in clear cell renal cell carcinoma via USP5-mediated HIF2α deubiquitination

doi: 10.1016/j.neo.2025.101186

Figure Lengend Snippet: pVHL mediates the ubiquitination and degradation of CYP1B1. A and B Assessment of CYP1B1 mRNA and protein levels in ACHN cells with stable VHL knockdown and 786O and OSRC2 cells with stable VHL overexpression via western blotting and RT‒qPCR. C Coimmunoprecipitation of ACHN cell whole-cell lysates with CYP1B1, pVHL or IgG antibodies, followed by western blotting with the respective antibodies. D 293T and 786O cells were transfected with Flag-CYP1B1 and His-pVHL plasmids. Coimmunoprecipitation with CYP1B1, pVHL or IgG antibodies was followed by western blotting with the indicated antibodies. E GST affinity-isolation assays with purified tagged pVHL and CYP1B1 proteins, followed by western blotting analysis. F Cotransfection of 293T and ACHN cells with VHL and CYP1B1 overexpression plasmids and subsequent immunofluorescence staining with anti-pVHL and anti-CYP1B1 antibodies. Scale bar: 10 μm. G Protein synthesis inhibition via CHX (10 μM) in 786O cells with VHL overexpression and ACHN cells with VHL knockdown; CYP1B1 protein levels were assessed by western blotting at specified time points. H and I Treatment of 786O and ACHN cells with VHL overexpression or knockdown plasmids, followed by exposure to DMSO, chloroquine (CQ, 10 μM), or MG132 (20 μM) for 8 hours; CYP1B1 protein levels were analysed by western blotting. J and K Immunoprecipitation of cells with anti-Flag CYP1B1 antibodies, followed by western blotting with the indicated antibodies (ns, not significant; **, p < 0.01).

Article Snippet: The OSRC2 cell line was acquired from ProCell (Wuhan, China) and maintained in RPMI-1640 medium enriched with 10 % FBS (Boster, Wuhan, China).

Techniques: Ubiquitin Proteomics, Knockdown, Over Expression, Western Blot, Transfection, Isolation, Purification, Cotransfection, Immunofluorescence, Staining, Inhibition, Immunoprecipitation

Journal: Neoplasia (New York, N.Y.)

Article Title: CYP1B1 promotes angiogenesis and sunitinib resistance in clear cell renal cell carcinoma via USP5-mediated HIF2α deubiquitination

doi: 10.1016/j.neo.2025.101186

Figure Lengend Snippet: CYP1B1 regulates HIF2α protein stability through the ubiquitin–proteasome pathway. A Immunoprecipitation of 293T and 786O cell lysates with CYP1B1 or HIF2α antibodies, followed by western blotting with the indicated antibodies. B The purified tagged proteins were subjected to GST pull-down assays with the indicated antibodies. C Confocal microscopy analysis of HIF2α and CYP1B1 colocalization in 786O and OSRC2 cells. Scale bar: 50 μm. D Protein synthesis inhibition in 786O cells overexpressing CYP1B1 and SU-R 786O cells with CYP1B1 knockdown via CHX (10 μM); HIF2α protein levels were determined by western blotting at specified time points. E and F Treatment of 786O cells overexpressing CYP1B1 and SU-R 786O cells with CYP1B1 knockdown with DMSO, chloroquine (10 μM), or MG132 (20 μM) for 8 hours; HIF2α protein levels were analysed by western blotting. G Immunoprecipitation of the indicated cell lysates with anti-Flag antibodies, followed by western blotting with the indicated antibodies. H Venn diagram illustrating potential deubiquitinating enzymes that regulate the degradation of ubiquitinated HIF2α. I Co-IP assays in 786O cells were performed to assess the interaction between the five deubiquitinating enzymes and HIF2α. J The purified tagged proteins were subjected to GST pull-down assays with the indicated antibodies (ns, not significant; **, p < 0.01; ***, p < 0.001).

Article Snippet: The OSRC2 cell line was acquired from ProCell (Wuhan, China) and maintained in RPMI-1640 medium enriched with 10 % FBS (Boster, Wuhan, China).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Purification, Confocal Microscopy, Inhibition, Knockdown, Co-Immunoprecipitation Assay