Journal: Oncotarget

Article Title: Expression and function of nuclear receptor coactivator 4 isoforms in transformed endometriotic and malignant ovarian cells

doi: 10.18632/oncotarget.23747

Figure Lengend Snippet: ( A ) Schematic depicting the overall strategy involving retroviral infections (with control virus (CV) or oncogenic cocktail virus (OCV: comprised of HRAS V12A , c-MYC T58A , SV40 LTAg, and HA-hTERT)) to generate transformed endometriotic from primary cells (PE-A, PE-B, PE-C, and PE-D; * refers to life-extended PE-D cells with SV40 LTAg) isolated from endometriotic lesions. Two batches of transformed endometriotic cells were successfully obtained using PE-A and PE-B primary cells. The first batch of retrovirally infected cells (PE-B-CV and PE-B-OCV) were utilized to: ( B ) obtain cell lysates for western blotting with the indicated antibodies (left panel). The dotted line specifies re-run samples to avoid the possibility of detecting overlapping bands of similar molecular weights. Densitometric analyses for pAKT and pMAPK are shown in the right panels; ( C ) perform colony formation assay and images were captured following 14 days in culture (representative images are shown, three independent experiments were conducted); ( D ) perform β-galactosidase staining and images were captured at 100× magnification (representative images are shown, three independent experiments were conducted); and ( E ) assess DNA damage via γH2AX immunofluorescence staining (representative images shown were captured at 63× magnification and the images of nuclei were enlarged and cropped using PowerPoint to focus on the DNA damage foci). The second batch of retrovirally infected cells (PE-B-CV and PE-B-OCV) were utilized to: ( F ) assess the in vitro tumorigenic potential (by 3-dimensional morphogenesis assay in Matrigel). Representative images (from four independent experiments) were captured at 100× (left) and 200× (right) magnification; ( G ) to measure IL-6 transcript levels via real-time PCR. Three independent experiments were performed; and ( H ) assess transcript levels for genes in the EMT pathway via real-time PCR (three independent experiments were performed).

Article Snippet: Oncogenic cocktail virus was generated using the following at equimolar quantities (a) H-RAS V12A (Addgene plasmid #9051, a gift from William Hahn), (b) c-MYC T58A (Addgene plasmid #20076, a gift from Juan Belmonte [ ]), (c) SV40 LTAg (Addgene plasmid #14088, a gift from William Hahn [ ]), and (d) HA-tagged hTERT (Addgene plasmid #1772, a gift from Bob Weinberg [ ]).

Techniques: Retroviral, Control, Virus, Transformation Assay, Isolation, Infection, Western Blot, Colony Assay, Staining, Immunofluorescence, In Vitro, Real-time Polymerase Chain Reaction