Journal: bioRxiv

Article Title: MafK mediates chromatin remodeling to silence IRF8 expression in non-immune cells in a lineage-specific manner

doi: 10.1101/396291

Figure Lengend Snippet: MafK25, MafK20 and MafK-int6 regulatory-regions (ai,bi,ci respectively) were divided into segments and MARE sites are illustrated in triangular (MafK) and rectangular (MafK/Bach1) with their predictive affinity score, as determined by the Jasper motif discovery . NIH3T3 were infected with pMSCV-IRF8p-Luc construct harboring either MafK25, MafK20 or MafK-int6 segmented regulatory-regions (aii,bii,cii respectively). Retroviral Luciferase activity was normalized to vector genomic copy number, as determined using real-time qPCR. Relative Luciferase activity was calculated as ratio between MafK construct activity and empty-vector (control). *p-value<0.05, **p-value<0.01 student’s t-test, n=2-3.

Article Snippet: The following antibodies were used: αH3K27ac (Abcam, CAT#ab4729), αH3K27me3 (Upstate, CAT#17-622), αMafK (Santa Cruz, CAT#sc-22831 and anti-normal rabbit IgG (Upstate, CAT#17-622), αBach1 (C-20) (Santa Cruz, CAT#sc14700x).

Techniques: Infection, Construct, Retroviral, Luciferase, Activity Assay, Plasmid Preparation, Control

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

Journal: Experimental & Molecular Medicine

Article Title: Generation of an osteoblast-based artificial niche that supports in vitro B lymphopoiesis

doi: 10.1038/emm.2017.189

Figure Lengend Snippet: Development of the OBN4 clone. ( a ) Schematic diagram of the SV40 Tag expression plasmid. FLAG-epitope (hatched box); LTR, retroviral long terminal repeat; Puro R , puromycin resistant cassette. ( b ) Expression of FLAG-tagged SV40 Tag in OBN cells. The expression of SV40 Tag was analyzed by western blotting with an anti-FLAG antibody. β-Actin was used as a loading control. pOB, primary osteoblast. ( c ) Comparison of ALP activities in OBN clones. The ALP activity levels of single OBN clones were analyzed through stimulation with 50 μ M ascorbic acid, 10 m M β-glycerophosphate and 10 −7 M dexamethasone for 6 d. * P <0.05. ** P <0.01.

Article Snippet: The collected cells were differentiated into primary osteoblasts by culture in α-MEM including 10% FBS and supplemented with 50 μ M ascorbic acid, 10 m M β-glycerophosphate, 100 ngml −1 BMP-2 and 10 −7 M dexamethasone for 5 d. The primary osteoblasts were further activated by treatment with 50 μ M ascorbic acid, 10 m M β-glycerophosphate and 10 −7 M PTH for 3 d. To prepare a retroviral supernatant harboring the SV40 Tag expression cassette, PlatE cells were transfected with retroviral SV40 Tag expression plasmid DNA using the TurboFect transfection reagent (Fermentas, Hanover, MD) according to the manufacturer's instructions.

Techniques: Expressing, Plasmid Preparation, FLAG-tag, Retroviral, Western Blot, Control, Comparison, Clone Assay, Activity Assay