Journal: mBio

Article Title: Characterization of Host and Microbial Determinants in Individuals with Latent Tuberculosis Infection Using a Human Granuloma Model

doi: 10.1128/mBio.02537-14

Figure Lengend Snippet: In vitro granuloma-like structures are formed by M. tuberculosis infection of PBMCs from LTBI individuals. (a) PBMCs obtained from LTBI individuals were infected with M. tuberculosis H 37 R v (MOI 1:1), resulting in the formation of granulomas by day 7 postinfection. (b) Higher magnification of the granulomas by confocal microscopy. (c) No formation of granulomas was observed in uninfected PBMCs obtained from LTBI individuals for up to 12 days postinfection. (d to f) Confocal microscopy images of the granulomas at day 7 postinfection revealed multicellular, multilayered structures containing approximately 4 to 8 cell layers. (d) Differential inference contrast (DIC) image. (e) Image of transverse and straight sections by orthogonal view. (f) Three-dimensional (3D) view image. Nuclei were stained with DAPI (cyan). (g) Granuloma-like structures include macrophages (CD11b + , red) and T cells (CD3 + , green). (h) Confocal microscopy images of the granulomas at day 7 postinfection revealed the presence of multinucleated giant cells (CD11b ++ , yellow; white arrow). Nuclei were stained with DAPI (dark blue). Representative images from n = 12 experiments are shown. The images in panels a and c are shown with ×40 magnification; the remaining images are ×60.

Article Snippet: The nuclei were stained with 0.1 μg/ml DAPI DNA stain (Molecular Probes, Carlsbad, CA).

Techniques: In Vitro, Infection, Confocal Microscopy, Staining

Journal:

Article Title: The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

doi: 10.1038/sj.emboj.7600252

Figure Lengend Snippet: Rps15p-depleted cells accumulate precursors to the 18S rRNA in the nucleus. (A) Pre-18S rRNA FISH with a probe complementary to the D–A2 segment of the ITS1 in wild-type, GAL-RPS15, GAL-RPS18, and GAL-RPS0 strains grown for 4 h in glucose-containing medium. Arrowheads indicate the nucleoplasm as visualized by DNA staining with DAPI. (B) Pre-5.8/25S rRNA detected with a probe to the E-C2 domain in the ITS2. (C) Growth of wild-type, GAL-RPS15, and GAL RPS18 cells. At t=4.5 h (dotted line), YP-galactose medium was replaced by YP-glucose. (D) Detection by electron microscopy of pre-18S RNAs with a riboprobe complementary to the D–A2 fragment of the ITS1. Cells were grown as in (A). Gold particles are highlighted in red. No: nucleolus; Np: nucleoplasm; Cy: cytoplasm. Bar=200 nm.

Article Snippet: Fluorescent detection was achieved with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) antibodies (Molecular Probes) and DNA was counterstained with DAPI.

Techniques: Staining, Electron Microscopy

Journal:

Article Title: The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

doi: 10.1038/sj.emboj.7600252

Figure Lengend Snippet: Behaviour of pre-ribosomal factors in Rps15p-depleted cells. (A) Immunolocalization of TAP-tagged Noc4p, Enp1p, Tsr1p, and Rio2p in wild-type and GAL-RPS15 strains cultured for 4 h in the presence of glucose. Immunofluorescence with anti-protein A antibodies. Arrowheads indicate the nucleoplasm as visualized by DNA staining with DAPI. (B) Detection of pre-18S rRNAs co-purifying with TAP-tagged Noc4p, Enp1p, Rio2p, and Tsr1p in Rps15p-depleted cells. GAL-RPS15 cells expressing one of the TAP-tagged proteins were grown for 4 h in YP-glucose medium and total cell extracts were submitted to immunoprecipitation with IgG-Sepharose. Co-precipitating RNAs were revealed after Northern blot with a probe hybridizing between points D and A2 in the ITS1. All samples, except Tsr1-TAP, were processed in parallel, starting with equivalent amounts of cell extract; one representative input lane (0.05% of input) is displayed. Semiquantification of the fraction of precipitated RNA was performed by phosphorimager analysis.

Article Snippet: Fluorescent detection was achieved with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) antibodies (Molecular Probes) and DNA was counterstained with DAPI.

Techniques: Cell Culture, Immunofluorescence, Staining, Expressing, Immunoprecipitation, Northern Blot