Journal: The Journal of Biological Chemistry

Article Title: Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner *

doi: 10.1074/jbc.M114.614719

Figure Lengend Snippet: Effect of integrin receptor ligation by ICAM-1 on CD16.NK-92 adhesion and cytolytic granule release. CD16.NK-92 cells were exposed to bilayers containing anti-CD16 antibody (50 mol/μm2) in the presence or absence of ICAM-1. A, percent of CD16.NK-92 cells that formed contacts with the bilayers (empty bars) and percent of the cells that released granules (slashed bars) are shown. The contact formation was determined as apparent change in the cell shape in transmitted light and by accumulation of fluorescent anti-CD16 antibodies at the cell/bilayer interface by TIRF microscopy. Granule release was detected by the appearance of CD107a molecules at the cell/bilayer interface by TIRF microscopy. The data are pooled average results from 3 independent experiments with 10 observation fields in each and are shown as mean ± S.D.; statistically significant differences were observed for both adherent and degranulating cells with p < 0.0001 by two-tailed Student's t test. B, representative images of CD16.NK-92 cells adhered to anti-CD16-containing bilayers with (left panel) or without (right panel) ICAM-1 molecules. Transmitted light images are overlaid with IRM images of the same cell. Dark areas correspond to tight contact between the cells and bilayers. C, average areas of tight contact formed by CD16.NK-92 cells exposed to the bilayers with (slashed bars) or without (empty bars) ICAM-1 are shown. Regions were drawn around tight adhesion areas on the IRM images, and areas of those regions were determined. Representative results of three independent experiments with at least 20 cells analyzed in each group; the data are shown as mean ± S.D., p < 0.0001 by two-tailed Student's t test.

Article Snippet: Images of CD16 clusters bound to fluorescent anti-CD16 antibodies were acquired with a TIRF microscope (Andor Revolution XD system equipped with Nikon TIRF-E illuminator and 100×/1.49 NA objective, Yokogawa CSU-X1 spinning disk confocal, and Andor iXon X3 EM-CCD camera).

Techniques: Ligation, Microscopy, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SARM1, not MyD88, mediates TLR7/TLR9-induced apoptosis in neurons 1

doi: 10.4049/jimmunol.1500953

Figure Lengend Snippet: Primary cortical neurons were stimulated with TLR7 and TLR9 ligands or mock controls. (A–E) Cells stained with HSP90 (green fluorescence) to identify neuronal cell delimitations and SARM1 (red fluorescence). SARM1 was observed primarily in the cell bodies (yellow dual fluorescence), but not the neurites of (A) mock-infected cells. However, (B–E) TLR stimulated neurons generally showed retracted neurites with focal areas of increased SARM1 (white arrows). (C–E) Close up showing punctate staining of SARM1 in neurites with(C) dual staining, (D) HSP90 and (E) SARM1. Images are representative of 2–3 repeated experiments. (F) Mitochondria were isolated from neurons at 72 hps and examined for the amount of SARM1 protein by western blot. VDAC1 was used as a loading control. (G–H) Quantification of SARM1 protein in (G) mitochondrial fraction normalized to VDAC1 expression as well as (H) whole cell lysates normalized to β-actin expression. Data are shown as fold change relative to mock controls. Error bars represent the range of 2 samples per group.

Article Snippet: Sections were blocked (5% BSA, 0.05% Triton in PBS) at room temperature for 30 min. Primary antibodies against GFP (antibody recognizes YFP protein, 1:500, Ab cam), Caspase 3 (1:250, Promega), SARM1 (1:100, ProSci) or TOM70 (1:150, Sigma-Aldrich) were incubated overnight at 4°C in PBS.

Techniques: Staining, Fluorescence, Infection, Isolation, Western Blot, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SARM1, not MyD88, mediates TLR7/TLR9-induced apoptosis in neurons 1

doi: 10.4049/jimmunol.1500953

Figure Lengend Snippet: (A–C) Primary cortical neurons from wildtype (C57BL/6) or Sarm1 −/− mice were stimulated with (A) TLR7/TLR9agonists (B) Kainic acid or (C) NMDA and analyzed for cell death at (A) 72 hps or (B–C) 18 hps. (A) Wildtype, but not SARM1−/− neurons underwent apoptosis following TLR7/TLR9 stimulation.. Data are the mean ± SD for 4–12 samples per group and are representative of 2–3 replicate experiments. (B–C) Wildtype and SARM1−/− neurons were treated with two-fold dilutions of (B) kainic acid or (C) NMDA. No statistical difference was observed between wildtype and SARM1−/− neurons for either drug. Data are the mean ± SD for 6 samples per group per concentration. Statistical analysis was completed with a two-way ANOVA with a Bonferroni’s multiple comparison post-test. *** P<0.001.

Article Snippet: Sections were blocked (5% BSA, 0.05% Triton in PBS) at room temperature for 30 min. Primary antibodies against GFP (antibody recognizes YFP protein, 1:500, Ab cam), Caspase 3 (1:250, Promega), SARM1 (1:100, ProSci) or TOM70 (1:150, Sigma-Aldrich) were incubated overnight at 4°C in PBS.

Techniques: Concentration Assay, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SARM1, not MyD88, mediates TLR7/TLR9-induced apoptosis in neurons 1

doi: 10.4049/jimmunol.1500953

Figure Lengend Snippet: Primary cortical neurons from (A–B) wildtype or (C–D) Sarm1−/− mice were stimulated with (B,D) TLR7/TLR9 ligands or (A,C) mock-stimulated. At 60 hps, neurons were incubated with MitoTracker Red CMXRos dye (red fluorescence) to detect mitochondria and Hoechst 33342 dye (blue fluorescence) as a counterstain. (B) white arrows show increased mitochondria (red fluorescence) in the neurites of neurons in TLR7/TLR9 stimulated cultures compared to (A) mock-treated cultures. (D) This increase of mitochondria in neurites was not observed in TLR7/TLR9-stimulated Sarm1−/− neurons. Images are representative of cultures for each group. (E) Quantification of mitotracker intensity in neurites. Red fluorescence intensity per every 100 nm was measured in neurites from mock and TLR7/TLR9-stimulated wildtype and SARM1−/− neurons. Data are the mean ± SD for 76–187 individual data points per group and include measurements from multiple neurites per image and multiple images per group. Statistical analysis was completed using One-way ANOVA with a Tukey’s multiple comparison test between all groups. *** P<0.0001

Article Snippet: Sections were blocked (5% BSA, 0.05% Triton in PBS) at room temperature for 30 min. Primary antibodies against GFP (antibody recognizes YFP protein, 1:500, Ab cam), Caspase 3 (1:250, Promega), SARM1 (1:100, ProSci) or TOM70 (1:150, Sigma-Aldrich) were incubated overnight at 4°C in PBS.

Techniques: Incubation, Fluorescence, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SARM1, not MyD88, mediates TLR7/TLR9-induced apoptosis in neurons 1

doi: 10.4049/jimmunol.1500953

Figure Lengend Snippet: Six week old OMP-YFP mice were administered intranasally (A,C, E) PBS or (B,D, E) PBS containing 25 μg of imiquimod and 0.5 μg of CpG-ODN 1826. At 72 hps, tissues were removed and processed for immunohistochemistry. (A–F) Nasal epithelium from (A, C, E) mock or (B, D, F) TLR agonist-inoculated mice were stained with (A–D) anti-GFP to detect OSNs (green fluorescence) and either (A–B) anti-active caspase 3 or (C–F) SARM1. Inset in (B) shows OMP-YFP positive OSN that is also positive for Caspase 3. (E–F) Only fluorescence of SARM1 is shown which demonstrates increase in SARM1 staining in OSN layer and axonal bundles (yellow outline) of TLR-stimulated mice. Blue arrows indicate SARM1 staining of OSN projections. All error bars are 30 um.

Article Snippet: Sections were blocked (5% BSA, 0.05% Triton in PBS) at room temperature for 30 min. Primary antibodies against GFP (antibody recognizes YFP protein, 1:500, Ab cam), Caspase 3 (1:250, Promega), SARM1 (1:100, ProSci) or TOM70 (1:150, Sigma-Aldrich) were incubated overnight at 4°C in PBS.

Techniques: Immunohistochemistry, Staining, Fluorescence

Journal: The EMBO Journal

Article Title: The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature

doi: 10.1038/s44318-024-00268-1

Figure Lengend Snippet: DsRed-CLC were co-expressed with Nedd4L-GFP in wild-type HeLa cells. Images were acquired at 1 s intervals through TIRF microscopy. Upper panel, TIRF images of HeLa cells. Insets represent enlarged images of the dashed boxes. Bottom panel, selected snapshots (every 10 s) from a time series were obtained from a single CCP indicated by the arrowhead. The time point at which Nedd4L started to accumulate was set 0. Scale bar, 2 μm. .

Article Snippet: Images were acquired for 5 min at 1 s intervals with a TIRF microscopy system (Olympus), a 100× oil immersion objective NA = 1.45 (Olympus) and MetaMorph Imaging System Software.

Techniques: Microscopy