ovcar3 cells  (ATCC)

Journal: Cells

Article Title: The USP19-DnaJC7 Axis Stabilizes p53 in Cisplatin-Treated Epithelial Ovarian Cancer

doi: 10.3390/cells15100925

Figure Lengend Snippet: DnaJC7, a putative binding partner of USP19, binds with USP19. ( A ) The putative binding partner nodes of USP19 are connected to DnaJC7, and those of DnaJC7 are connected to p53. Both DnaJC7 and USP19 nodes are colored yellow. The visualization is generated using Cytoscape version 3.9 with STRING network, and the thickness of edges represents the interaction score between protein nodes. ( B ) Binding assays between USP19 and DnaJC7 are performed. Myc-USP19 and Flag-DnaJC7 are overexpressed in HEK293T cells, followed by IP using Flag and Myc antibodies, respectively. ( C ) GST pull-down assay is conducted to confirm the binding between DnaJC7 and USP19. Myc-USP19 overexpressed in HEK293T cells is incubated with purified GST-DnaJC7. ( D ) ICC assay is conducted to confirm the co-localization of DnaJC7 and USP19 in various EOC cell lines (A2780, OVCAR3, and SKOV3). A mouse-host DnaJC7 antibody and a rabbit-host USP19 antibody are utilized. 4′,6-diamidino-2-phenylindole DAPI is used to stain the nucleus. Scale bars represent 20 µm. ( E ) To determine which domain of DnaJC7 binds to USP19, domain mutant constructs of DnaJC7 are generated and a binding assay with Myc-USP19 is conducted in HEK293T cells. IP is performed using a Myc antibody, followed by detection using a USP19 antibody. ( F ) To confirm the binding between the domains of DnaJC7 and USP19, USP19 domain constructs are utilized in HEK293T cells. A mouse-host DnaJC7 antibody is used for IP, and a rabbit-host DnaJC7 antibody is used for WB.

Article Snippet: A2780 (RRID: CVCL_0134; T8089, Applied Biological Materials Inc., Richmond, BC, Canada) and OVCAR3 (RRID: CVCL_0465; HTB-161, ATCC) cells were grown in Roswell Park Memorial Institute (RPMI; 11875-093, Gibco) medium.

Techniques: Binding Assay, Generated, Pull Down Assay, Incubation, Purification, Immunocytochemistry, Staining, Mutagenesis, Construct

Journal: Cells

Article Title: The USP19-DnaJC7 Axis Stabilizes p53 in Cisplatin-Treated Epithelial Ovarian Cancer

doi: 10.3390/cells15100925

Figure Lengend Snippet: DnaJC7 upregulates the stability of p53 by inhibiting ubiquitination. ( A – C ) To confirm the association between DnaJC7 and the stability of p53 (WT, R175H, and R248Q), the expression of p53 is assessed in A2780 (WT), TOV-112D (R175H), and OVCAR3 (R248Q) cells overexpressing Flag-DnaJC7 in a dose-dependent manner. The graph on the right displays the mean and SD of p53 expression levels from repeated experiments (n = 3). Statistical analysis is conducted using one-way ANOVA. ( D ) To examine the polyubiquitination of p53 based on the expression levels of DnaJC7 and USP19, Flag-DnaJC7, Myc-USP19, and HA-Ub are overexpressed in HEK293T cells. Rabbit-host primary p53 antibodies are used for WB, whereas mouse-host primary p53 antibodies are employed for IP. ( E ) To identify the regulation of p53 ubiquitination through the USP19-DnaJC7 axis, Myc- USP19 and siDnaJC7 are used in HEK293T cells. ( F ) Binding of p53 and DnaJC7 is confirmed by incubating purified GST-DnaJC7 with HEK293T cell lysates overexpressing Myc-p53. ( G ) The binding of MDM2 and DnaJC7 is assessed by incubating purified GST-DnaJC7 with HEK293T cell lysates overexpressing Flag-MDM2. Significant p -values are indicated with asterisks (ns = not significant, * p < 0.05, and ** p < 0.01).

Article Snippet: A2780 (RRID: CVCL_0134; T8089, Applied Biological Materials Inc., Richmond, BC, Canada) and OVCAR3 (RRID: CVCL_0465; HTB-161, ATCC) cells were grown in Roswell Park Memorial Institute (RPMI; 11875-093, Gibco) medium.

Techniques: Ubiquitin Proteomics, Expressing, Binding Assay, Purification