Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Nonstructural Membrane Protein pK15 Recruits the Class II Phosphatidylinositol 3-Kinase PI3K-C2α To Activate Productive Viral Replication

doi: 10.1128/JVI.00544-18

Figure Lengend Snippet: Depletion of PI3K-C2α from infected endothelial cells leads to reduced KSHV lytic replication. HuARLT2-rKSHV cells were microporated with either a control siRNA (Scr), four individual siRNAs or a pool of these siRNAs against PI3K-C2α, or a single siRNA against K15, and the KSHV lytic cycle was induced 24 h later using a cocktail of RTA and SB. (A to C) Forty-eight hours after lytic induction, images for GFP and RFP expression were acquired (A), and the number of RFP-positive cells from five or more fields was quantified; cells were then lysed, and the expression level of the indicated viral proteins was assessed by Western blotting (C). Results are representative of data from two independent experiments. The scatter plot (B) represents the means ± standard deviations (SD) for five or more fields. Ordinary one-way analysis of variance (ANOVA) was used to determine P values. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D and E) Primary lymphatic endothelial cells infected with KSHV for 2 weeks (LEC-rKSHV) were transfected with either the pooled PI3K-C2α siRNA or a scrambled control. After 72 h, cells and the culture supernatant were collected. The expression levels of the indicated proteins were analyzed by Western blotting (D), and the amount of released infectious virus was assayed by titrating the culture supernatant on HEK-293 cells (E). A Mann-Whitney test was used to determine P values. **, P < 0.01.

Article Snippet: Primary juvenile foreskin lymphatic endothelial cells (LECs; Lonza) were provided by Päivi M. Ojala (University of Helsinki, Helsinki, Finland) and were grown in EGM-2MV medium (Lonza, Walkersville, MD, USA).

Techniques: Infection, Control, Expressing, Western Blot, Transfection, Virus, MANN-WHITNEY

Journal: Epigenetics & Chromatin

Article Title: CGGBP1 regulates CTCF occupancy at repeats

doi: 10.1186/s13072-019-0305-6

Figure Lengend Snippet: CGGBP1 and CTCF colocalize in the nucleus. a Human juvenile fibroblasts were co-immunostained for CGGBP1 (green) and CTCF (red). Nuclei were counterstained with DAPI (blue). b Zoomed view of a representative nucleus (630× zoom, focal plane thickness 0.896 micrometers) shows some overlap between signals for DAPI, CGGBP1 and CTCF. c Blocking was done with anti-CGGBP1 rabbit polyclonal antibody followed by incubation with detection antibody (anti-CGGBP1 rabbit polyclonal antibody and anti-CTCF mouse monoclonal antibody). d Blocking was done with anti-CTCF mouse monoclonal antibody followed by incubation with detection antibody (anti-CGGBP1 rabbit polyclonal antibody and anti-CTCF mouse monoclonal antibody). Micrographs show signal for DAPI (blue), CGGBP1 (green) and CTCF (red). e PLA (red foci) shows the proximity between CTCF and CGGBP1 in situ. Nuclei were stained with DAPI (blue). CGGBP1–CTCF proximity was stronger in the nuclei than cytoplasm (inset of rabbit anti-CGGBP1:mouse anti-CTCF sample). No significant PLA signal was observed in IgG and negative controls with no-primary antibody (inset of no-primary antibody sample). f Counting of PLA signals shows proximity detected using specific antibodies as significantly higher than the negative controls. Also the PLA signal frequency in the nuclei (blue) was higher than in the cytoplasm (green). All images were captured with confocal planes of 1.601 µm

Article Snippet: Human dermal fibroblast (Sigma, passage 15–24), human juvenile foreskin fibroblast (Himedia, passage 5–30) and HEK-293T cells were grown in DMEM (AL007A) supplemented with 10% FBS.

Techniques: Blocking Assay, Incubation, In Situ, Staining