Journal: Journal of Lipid Research

Article Title: Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake

doi: 10.1194/jlr.M041335

Figure Lengend Snippet: miR-185 downregulates SREBP-2 mRNA and protein level by binding to MREs within SREBP-2 mRNA 3′UTR. A: Luciferase activity was quantitated in 293T cells transfected with a control luciferase reporter plasmid (Con Luc), SREBP-2 3′UTR containing reporter plasmid (3′UTR), SREBP-2 3′UTR mutant (3′UTR MUT), and pre-miR-185 (miR185) or control pre-miR (Con miR). Luciferase activity was measured in 293T cells as described in the Materials and Methods section. Bar graphs represent mean ± SEM from three independent experiments. B: The fold change in SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185, Antagamer-miR-185 (Antagomer) (100 nM), or control miR (Con miR) transfected HepG2 cells. Bar graphs represent mean ± SEM from three independent experiments. C: SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185 or control miR transfected THLE-2 cells. Values were normalized to the level of GAPDH. Bar graphs represent mean ± SEM from three independent experiments. D: SREBP-2 protein level was determined using whole cell lysates by Western analysis in miR-185 overexpressing and control miR transfected HepG2 cells. β-Actin was used as a loading control. FL, full-length SREBP-2; M, mature form of SREBP-2.

Article Snippet: THLE-2, HepG2, and 293T cells were obtained from ATCC.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS Pathogens

Article Title: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

doi: 10.1371/journal.ppat.1003971

Figure Lengend Snippet: MDCK cells were infected with wild type (WT) WSN at a multiplicity of infection (MOI) of 3 for 8 hours post infection (hpi) and then imaged with FISH probes directed against four distinct vRNA species. The first row displays a cell probed for influenza vRNA segments PB2, PB1, PA, and NP. Row 3 contains images of a cell probed for the other 4 influenza vRNA segments: HA, NA, M and NS. The second and fourth row of images show an enlargement of the area defined by the dashed boxes in the first and third rows. Solid white arrowheads identify a cytoplasmic focus with all four distinct vRNA segments, turquoise arrowheads indicate a focus with only three vRNA species, and open arrowheads show a focus with only two vRNA species. All scale bars are 10 µm. DAPI marks the cellular nucleus.

Article Snippet: Madin-Darby Canine kidney (MDCK) epithelial cells and adenocarcinoma human alveolar basal epithelial cells (A549) obtained from ATCC were maintained in modified Eagle's medium (MEM) with 10% fetal calf serum (FCS) and L-glutamine or Dulbecco's MEM (DMEM) with 10% FCS respectively.

Techniques: Infection

Journal: PLoS Pathogens

Article Title: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

doi: 10.1371/journal.ppat.1003971

Figure Lengend Snippet: 3D rendering of an MDCK cell infected with WT WSN (MOI = 3) for 8 hpi were stained with probe B from : WSN PB2 Quasar 570 (red spots), PB1 Alexa-Fluor 488 (green spots), PA Cal Fluor Red 590 (orange spots), and NP Quasar 670 (yellow spots) (A). The DAPI-stained nucleus is labeled in blue. An enlarged region of the 3D image was tilted 90° in the z-direction to provide an axial-view of the cytoplasmic foci and nucleus. Scale bars are 4 µm in the whole cell image and 2 µm in the rotated images. The total number of foci containing 1, 2, 3, or 4 vRNA segments was quantified within a given cell (B). Each bar represents the average from 3 independently analyzed cells with standard error indicated. The distance from the nucleus for each focus from four independent cells was calculated (C). The proportion of foci containing 4,3,2,or 1 distinct vRNA segment with a given range from the nucleus is represented graphically as a scatter plot. Each spot is an average from 4 independent cells and the standard error is indicated.

Article Snippet: Madin-Darby Canine kidney (MDCK) epithelial cells and adenocarcinoma human alveolar basal epithelial cells (A549) obtained from ATCC were maintained in modified Eagle's medium (MEM) with 10% fetal calf serum (FCS) and L-glutamine or Dulbecco's MEM (DMEM) with 10% FCS respectively.

Techniques: Infection, Staining, Labeling

Journal: PLoS Pathogens

Article Title: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

doi: 10.1371/journal.ppat.1003971

Figure Lengend Snippet: Schematic of the reverse genetics pHW2000 plasmid created by inserting the coding region of full length GFP into the C-terminal domain of the WSN PA open reading frame (A). This insertion included a duplication of 162 nucleotides of WSN PA prior to the 5′ non-coding region (NCR) depicted in the black rectangle. Western blot of purified recombinant WT WSN or WSN PA-GFP virus grown in embryonated eggs (B). The GFP signal corresponds to the appropriate size of a PA-GFP fusion protein. Viral NP protein was used as a control to ensure loading of equivalent amounts of virus. Immunofluorescence of MDCK cells infected with WSN PA-GFP (MOI = 3) for 16 hpi with anti-influenza NP antibody (C). Images to the right are enlarged regions identified by the dashed square. White arrows show areas of co-localization. All scale bars are 5 µm.

Article Snippet: Madin-Darby Canine kidney (MDCK) epithelial cells and adenocarcinoma human alveolar basal epithelial cells (A549) obtained from ATCC were maintained in modified Eagle's medium (MEM) with 10% fetal calf serum (FCS) and L-glutamine or Dulbecco's MEM (DMEM) with 10% FCS respectively.

Techniques: Plasmid Preparation, Western Blot, Purification, Recombinant, Virus, Control, Immunofluorescence, Infection

Journal: PLoS Pathogens

Article Title: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

doi: 10.1371/journal.ppat.1003971

Figure Lengend Snippet: Fluorescent in situ hybridization (FISH) for viral RNA segments 1 (PB2) and 4 (HA) on MDCK (A) or A549 cells (B) cells 16 hpi with WSN PA-GFP (MOI = 0.5). Images to the right are enlarged regions identified by the dashed box. White arrowheads show colocalization of PA-GFP and vRNA, and open arrowheads indicate GFP foci not colocalized with viral RNA signal. DAPI marks the cellular nucleus. Scale bars are 5 µm in all panels of part A. In part B scale bars are 10 µm in the whole cell images and 2 µm in length in the enlarged region.

Article Snippet: Madin-Darby Canine kidney (MDCK) epithelial cells and adenocarcinoma human alveolar basal epithelial cells (A549) obtained from ATCC were maintained in modified Eagle's medium (MEM) with 10% fetal calf serum (FCS) and L-glutamine or Dulbecco's MEM (DMEM) with 10% FCS respectively.

Techniques: In Situ Hybridization

Journal: PLoS Pathogens

Article Title: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

doi: 10.1371/journal.ppat.1003971

Figure Lengend Snippet: Maximum intensity projection from inverted selective-plane illumination microscopy (iSPIM) movie of MDCK cells infected with WSN PA-GFP (MOI = 5) at 16 hpi, 36 seconds (s) after onset of tracking (A). The red-boxed region is highlighted in (B and C), and the red star indicates the region where colocalization of two GFP foci (marked in red and blue arrows) occurred. A single WSN PA-GFP focus trajectory; each red dot is a spot tracked in time, the yellow line is the overall trace, and white arrows indicate the direction of the PA-GFP movement (B). The total track duration is 46 s. Part C contains still frames of the red-boxed region in part A from 26–44 s, and illustrates colocalization and subsequent fusion of two foci. The red arrow indicates the PA-GFP focus that is being tracked and the blue arrow indicates the PA-GFP focus with which it fuses. All scale bars are 2 µm. An intensity versus depth plot of the colocalized foci, indicated by the red star in part A, demonstrates a single peak indicating that the fusion occurs in a single diffraction-limited spot (D). An intensity vs. time plot of the fused focus compared to an adjacent stationary focus, identified by the white circle in part C, during the same time frame (E). The asterisk marks the time corresponding to the colocalization event.

Article Snippet: Madin-Darby Canine kidney (MDCK) epithelial cells and adenocarcinoma human alveolar basal epithelial cells (A549) obtained from ATCC were maintained in modified Eagle's medium (MEM) with 10% fetal calf serum (FCS) and L-glutamine or Dulbecco's MEM (DMEM) with 10% FCS respectively.

Techniques: Microscopy, Infection