Journal: Journal of Heredity

Article Title: Differences in Cell Proliferation and Craniofacial Phenotype of Closely Related Species in the Pupfish Genus Cyprinodon

doi: 10.1093/jhered/esz074

Figure Lengend Snippet: Immunofluorescence light sheet imaging visualizes number and location of mitotic cells as indicated by assay for pH3 in the heads of hatching-age Cyprinodon. (A) Regions of the head analyzed for proliferating cells. (B) 3D reconstructions in lateral view of the head for the DAPI channel (gray, stains nuclei), the pH3 channel (red, dividing cells), and both channels merged. Note that pH3 positive cells (red dots) are mainly localized to ventral structures as shown in lateral view. (C, D) Single 2D images in (C) frontal view and (D) lateral view show pH3 cells (red dots) localizing to epithelial and mesenchyme tissues surrounding cartilage elements as exemplified by the ceratohyal. Cartilage cells are identifiable in the DAPI channel (gray) by their widely spaced nuclei indicative of large cuboidal cells. Cartilage elements can be identified by shape, and are outlined by the brightly labeled perichondrium, a dense layer of mesenchyme cells that surrounds cartilage elements. Inset shows region outlined by white box and arrows point to pH3 positive cells in perichondrium. Note also the clusters of pH3 positive cells in jaws (arrow head), especially around lateral edge of jaws as seen in panel C. Labels: bh, basihyal cartilage; br, brain; ch, ceratohyal cartilage; ey, eye; ga, gill arches; lj, lower jaw; pa, pharynx; pf, pectoral fin; uj, upper jaw.

Article Snippet: Cells were labeled for pH3 with primary rabbit anti-pH3 polyclonal antibody (MyBioSource, MBS2517807), and secondary antibody Alexa Fluor 568 goat anti-Rabbit (Thermo Fisher, A11011).

Techniques: Immunofluorescence, Imaging, Labeling

Journal: Journal of Heredity

Article Title: Differences in Cell Proliferation and Craniofacial Phenotype of Closely Related Species in the Pupfish Genus Cyprinodon

doi: 10.1093/jhered/esz074

Figure Lengend Snippet: Cell proliferation varies among species of Cyprinodon in different regions of the head at hatching. Shown are number of pH3 positive cells relative to either surface area of sampled tissue (A) or volume of sampled tissue (B) for the 3 regions of the head sampled plus the index of relative jaw proliferation (see text). Plotted are values for each sample and boxplots. Samples sizes are snail-eater N = 30, omnivore N = 28, scale-biter N = 19. Significance for all post hoc pairwise comparisons (Tukey) is shown above boxplots and corresponds to Supplementary Table S3. Note that levels of proliferation significantly vary between species when sampling the entire head (e.g., head region standardized to surface area) or regions of the head posterior to the jaws (post-jaw subset), but proliferation does not vary in the jaws (jaw subset). ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Cells were labeled for pH3 with primary rabbit anti-pH3 polyclonal antibody (MyBioSource, MBS2517807), and secondary antibody Alexa Fluor 568 goat anti-Rabbit (Thermo Fisher, A11011).

Techniques: Sampling

Journal: PLoS ONE

Article Title: Snapin, Positive Regulator of Stimulation- Induced Ca 2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

doi: 10.1371/journal.pone.0075297

Figure Lengend Snippet: (A) NFAT Luc reporter plasmids were transfected into Jurkat cells expressing the indicated peptide with pBMN lacZ as the internal control plasmid. Cells were treated for 3 hr with or without indicated agents (2 µ g/ml PHA and 10 ng/ml PMA) prior to measurement of luciferase activity. The experiments were repeated three times; values shown are the average ± SE. Untreated Jurkat cells were assigned a value of 1 and data from these cells were used to calculate the fold activation. Transfection efficiencies were normalized to a co-transfected lacZ plasmid. (B)'pBMN-control IRES-Lyt2 α ' or pBMN-Snapin IRES-Lyt2 α ' retrovirus vectors were transduced into SupT1 cells expressing C-Pep1 or Pep80. These cells were challenged with HIV-1 (NL4-3) at a dose 400 TCID 50 per 5×10 4 cells. P24 gag levels in culture supernatants were assayed from four wells on the indicated days after infection. P24 gag levels were normalized to cell number determined using an XTT assay. Data are presented as the average ± SE per 10 6 cells. Similar results were observed in three independent experiments. (C) 293T cells were co-transfected with the indicated combinations of expression vectors: HA-Pep80, GST-p65 (control), or GST-Snapin. Cell lysates were immunoprecipitated with anti-HA mAb and immunoblotted with anti-GST mAb. Purified GST-p65 (control) and GST-Snapin are shown as controls and marked with asterisks.

Article Snippet: Snapin was detected using a primary rabbit polyclonal anti-Snapin antibody (148002, Synaptic Systems) and a DyLightTM488-conjugated secondary anti-rabbit (711-485-152, Jackson ImmunoResearch).

Techniques: Transfection, Expressing, Control, Plasmid Preparation, Luciferase, Activity Assay, Activation Assay, Infection, XTT Assay, Immunoprecipitation, Purification

Journal: PLoS ONE

Article Title: Snapin, Positive Regulator of Stimulation- Induced Ca 2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

doi: 10.1371/journal.pone.0075297

Figure Lengend Snippet: Jurkat cells were immunostained with anti-Snapin antibody and anti-calnexin antibody. Cells were then examined by confocal microscopy. Z; Reconstructed three-dimensional structures of Jurkat cells based on Z-stack analysis of immunostained Snapin (green) and calnexin (red). Nuclei were stained with DAPI (blue). White arrows indicate co-localization. Bar, 10 µ m.

Article Snippet: Snapin was detected using a primary rabbit polyclonal anti-Snapin antibody (148002, Synaptic Systems) and a DyLightTM488-conjugated secondary anti-rabbit (711-485-152, Jackson ImmunoResearch).

Techniques: Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Snapin, Positive Regulator of Stimulation- Induced Ca 2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

doi: 10.1371/journal.pone.0075297

Figure Lengend Snippet: After crosslinking the ER fraction from Jurkat cells and from C-Pep-1- or Pep80-expressing Jurkat cells, samples were immunopreciptated with anti-RyR3 antibody and immunoblotted with anti-Snapin antibody.

Article Snippet: Snapin was detected using a primary rabbit polyclonal anti-Snapin antibody (148002, Synaptic Systems) and a DyLightTM488-conjugated secondary anti-rabbit (711-485-152, Jackson ImmunoResearch).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Snapin, Positive Regulator of Stimulation- Induced Ca 2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

doi: 10.1371/journal.pone.0075297

Figure Lengend Snippet: (A, B) Indicated cells were suspended in Ca 2+ -free medium containing 10 mM EGTA. “Control” indicates control retrovirus, whereas “Snapin” indicates that cells were infected with Snapin-encoding retrovirus. Cells were transduced with either Pep80 or C-Pep1. Cells were stained with APC-anti-Lyt2 α ' and were loaded with indo-1-AM calcium sensor dye. EGTA was added, and after 30 s (A) OKT3 or (B) thapsigargin was added. The FL5/FL4 ratio (400 nm/510 nm fluorescence emission) was monitored using a flow cytometer. (C) Cells were suspended in medium containing Ca 2+ . After 30 s, OKT3 was added. The FL5/FL4 ratio was monitored using a flow cytometer.

Article Snippet: Snapin was detected using a primary rabbit polyclonal anti-Snapin antibody (148002, Synaptic Systems) and a DyLightTM488-conjugated secondary anti-rabbit (711-485-152, Jackson ImmunoResearch).

Techniques: Control, Infection, Transduction, Staining, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Snapin, Positive Regulator of Stimulation- Induced Ca 2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

doi: 10.1371/journal.pone.0075297

Figure Lengend Snippet: Jurkat cells that were transfected with Snapin-specific siRNA or control siRNA were suspended in (A) Ca 2+ -free medium with 10 mM EGTA or (B) Ca 2+ -containing medium. After 30 s, OKT3 was added. The FL5/FL4 ratio was monitored using a flow cytometer. (C) NFAT Luc reporter plasmid and pBMN lacZ were transfected into Jurkat cells that were transfected with Snapin-specific siRNA or control siRNA. Cells were treated for 3 hr with or without PHA and PMA prior to measurement of luciferase activity. The experiments were repeated three times; values shown are the average ± SE. Data from cells without treatment were assigned a value of 1 and were used to calculate the fold activation. Transfection efficiencies were normalized to a co-transfected lacZ plasmid. (D) SupT1 cells that were transfected with Snapin-specific siRNA or control siRNA were challenged with replication incompetent HIV-1-Ea. P24 gag levels in culture supernatants were assayed from four wells 48 hr after HIV-1 challenge. P24 gag levels were normalized for cell number using XTT assay. Data are presented as the average ± SE per 10 6 cells. Similar results were observed in three independent experiments. * indicates p <0.05 by t test.

Article Snippet: Snapin was detected using a primary rabbit polyclonal anti-Snapin antibody (148002, Synaptic Systems) and a DyLightTM488-conjugated secondary anti-rabbit (711-485-152, Jackson ImmunoResearch).

Techniques: Transfection, Control, Flow Cytometry, Plasmid Preparation, Luciferase, Activity Assay, Activation Assay, XTT Assay

Journal: PLoS ONE

Article Title: Snapin, Positive Regulator of Stimulation- Induced Ca 2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells

doi: 10.1371/journal.pone.0075297

Figure Lengend Snippet: pBMN-control-IRES-Lyt2 α ' and pBMN-Snapin-IRES-Lyt2α' retrovirus vectors were transduced into human primary CD4 + T cells. CD4 + T cells were challenged by HIV-1 (NL4-3) at 400 TCID 50 per 1×10 5 cells. P24 gag levels in culture supernatants were assayed from four wells on the indicated days after infection. P24 gag levels were normalized for cell number using an XTT assay. Data are presented as the average ± SE per 10 6 cells. Similar results were observed in three independent experiments. N.D.; not detected.

Article Snippet: Snapin was detected using a primary rabbit polyclonal anti-Snapin antibody (148002, Synaptic Systems) and a DyLightTM488-conjugated secondary anti-rabbit (711-485-152, Jackson ImmunoResearch).

Techniques: Control, Infection, XTT Assay