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: Nature immunology

Article Title: The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.

doi: 10.1038/ni1382

Figure Lengend Snippet: Figure 5 SARM associates with and inhibits TRIF signaling. (a) ELISA of RANTES production by HEK293 cells transfected for 24 h with SARM and TRIF expression plasmids. (b,c) Luciferase assay for Ccl5 promoter induction (b) or IRF3 activation (c) in HEK293 cells transfected for 24 h with SARM expression plasmid (amount (in ng), horizontal axes) and TRIF expression plasmid. (d) Luciferase assay for induction of the Ifnb promoter in HEK293 cells transfected as in b,c, but with 50 ng TRAM expression plasmid. (e) Luciferase assay for NF-kB activation in HEK293 cells transfected as in b,c, but with 50 ng RIP1 expression plasmid. (f) Luciferase assay for IRF3 activation in HEK293 cells transfected as in b,c, but with 50 ng TBK1. (g) Immunoprecipitation (IP) and immunoblot (IB) analysis of lysates of HEK-TLR4 cells transfected for 36 h with hemagglutinin-tagged TRIF (HA-TRIF) and Flag-tagged SARM. +LPS, cells treated for 15 min with 10 ng/ml of LPS; a-, antibody to. (h) Immunoprecipitation and immunoblot analysis of lysates of human PBMCs left unstimulated (0) or treated for 10 min with 100 ng/ml of LPS (10) and then lysed in extraction buffer. (i) Pairwise interactions assessed in yeast cells transformed with BD-SARM and AD-TRIF or AD-A46R and grown on ‘dropout’ agar plates (above lanes) lacking leucine (–Leu), tryptophan (–Trp), histidine (–His) and/or adenine (–Ade). Growth on middle and right plates indicates a direct interaction. Experiments are one of three repeats (error bars, s.d.).

Article Snippet: Antibody to SARM (anti-SARM) was from ProSci, monoclonal anti–FLAG M2 was from Sigma, and anti-hemagglutinin was from Covance.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Luciferase, Activation Assay, Plasmid Preparation, Immunoprecipitation, Western Blot, Extraction, Transformation Assay

Journal: Nature immunology

Article Title: The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.

doi: 10.1038/ni1382

Figure Lengend Snippet: Figure 6 The TIR and SAM domains of SARM are critical for TRIF inhibition. (a) Protein motifs of Flag-tagged SARM and truncated SARM constructs. S, SAM; amino acid positions, above diagrams. (b) Luciferase assay of HEK-TLR3 cells transfected for 24 h with the NF-kB reporter plus vector (V) or increasing amounts (wedges) of SARM expression plasmid or constructs (from a) expressing truncation proteins; cells were collected 6 h after stimulation with 25 mg/ml of poly(I:C). (c) Immunoblot of lystates of HEK-TLR4 cells transfected for 24 h with SARM or SARMDN expression plasmid and then treated with 100 ng/ml of LPS (time, above lanes). Experiments are one of three repeats (error bars, s.d.).

Article Snippet: Antibody to SARM (anti-SARM) was from ProSci, monoclonal anti–FLAG M2 was from Sigma, and anti-hemagglutinin was from Covance.

Techniques: Inhibition, Construct, Luciferase, Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Nature immunology

Article Title: The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.

doi: 10.1038/ni1382

Figure Lengend Snippet: Figure 7 ‘Knockdown’ of SARM expression enhances TRIF- but not MyD88- dependent gene induction. (a) RT-PCR of SARM1 mRNA ‘knockdown’ in HEK293 cells by the siRNA oligonucleotides S1 and S2 but not by nonspecific siRNA (N). –, cells not transfected with siRNA; Actb, loading control (b-actin mRNA). (b) Luciferase reporter assay for Ifnb promoter induction in lysates of HEK293 cells transfected with TRIF expression plasmid, Ifnb promoter reporter and either control or SARM1-specific siRNA simultaneously; after 24 h, the siRNA transfection was repeated, followed by analysis 24 h later. (c) Luciferase assay for Ifnb promoter induction by HEK-TLR3 cells transfected for 24 h with the Ifnb reporter and siRNA; the siRNA transfection was then repeated for a further 24 h, followed by stimulation for 24 h with 25 mg/ml of poly(I:C). (d) ELISA of RANTES (d) or IL-8 (e) in supernatants of HEK-TLR3 (d) or HEK293 (e) cells treated with siRNA 24 and 48 h after seeding, then stimulated for 24 h with either 25 mg/ml of poly(I:C) (d) or 100 ng/ml of IL-1a (e). (f) ELISA of RANTES (f) or TNF (g) in supernatants of PBMCs left untreated (–) or treated with siRNA 24 and 48 h after seeding, then stimulated for 24 h with either 25 mg/ml of poly(I:C) (f) or 100 ng/ml of LPS (g). Experiments are one of three repeats (error bars, s.d.).

Article Snippet: Antibody to SARM (anti-SARM) was from ProSci, monoclonal anti–FLAG M2 was from Sigma, and anti-hemagglutinin was from Covance.

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Luciferase, Reporter Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Innate Immunity Adaptor SARM Translocates to the Nucleus to Stabilize Lamins and Prevent DNA Fragmentation in Response to Pro-Apoptotic Signaling

doi: 10.1371/journal.pone.0070994

Figure Lengend Snippet: A. Design of SARM constructs used in this study. SARM's modular structure is comprised of ARM repeats (blue), a SAM domain (pink) and a TIR domain (orange). All SARM constructs contain an NH 2 -terminal AU1 tag (black) to facilitate immunoblotting of expressed proteins. CMV or LTR promoters are indicated. B. Fluorescent confocal images showing the localization patterns of transfected eGFP and eGFP fusion proteins in HEK 293 cells. Images were taken 24 h following transient transfection. White scale bars = 10 µm. C. Fluorescent confocal image of HEK293 cells fixed and permeabilized before staining with an antibody recognizing lamin A/C, visualized with a red fluorescent secondary antibody (see Materials and Methods for details). D. Propidium iodide (PI) counterstaining confirms nuclear localization of SARM-eGFP and ARM-eGFP and cytoplasmic localization of TIR-eGFP. Only full-length SARM-eGFP forms a lattice in the nucleus. Results shown are representative of three independent experiments.

Article Snippet: Immunoblotting of nuclear extracts was performed using a polyclonal anti-SARM antibody (United States Biological) recognizing the C-terminus of SARM and a polyclonal antibody to lamin A/C (Abcam) that recognizes both full-length lamin A and C (∼70 kDa) and a 28 kDa lamin cleavage fragment.

Techniques: Construct, Western Blot, Transfection, Staining

Journal: PLoS ONE

Article Title: The Innate Immunity Adaptor SARM Translocates to the Nucleus to Stabilize Lamins and Prevent DNA Fragmentation in Response to Pro-Apoptotic Signaling

doi: 10.1371/journal.pone.0070994

Figure Lengend Snippet: HEK 293 cells stably transfected with SARM-Neo or empty Neo vector were stimulated with TNF-α and Actinomycin-D (Act-D) for the times indicated to induce apoptosis, then analyzed for lamin degradation, DNA fragmentation and caspase 6 activity. Gel segments shown in each panel represent sections of a single gel. In A and B, β-actin is shown as a loading control. A. Transfected cells were left unstimulated or stimulated for 14 h. Nuclear extracts were prepared and analyzed by immunoblotting with anti-SARM to confirm SARM expression. B. Immunoblot analysis of nuclear extracts shows a reduction of the lamin cleavage fragment in SARM-expressing cells after 14 h stimulation. C. Intrachromosomal DNA fragmentation (200 base pair-spaced “ladder” banding pattern) is almost completely inhibited in SARM-expressing cells after 14 h stimulation. Equal amounts of total cell DNA were loaded in each well of an agarose gel. D. Caspase 6 activity is not affected by SARM expression in cells stimulated for 14 h. Results shown in A–C are representative of three independent experiments. Data in D are shown as the mean + SD of three independent experiments performed in triplicate (*** p <0.0001; NS = not significant).

Article Snippet: Immunoblotting of nuclear extracts was performed using a polyclonal anti-SARM antibody (United States Biological) recognizing the C-terminus of SARM and a polyclonal antibody to lamin A/C (Abcam) that recognizes both full-length lamin A and C (∼70 kDa) and a 28 kDa lamin cleavage fragment.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Activity Assay, Control, Western Blot, Expressing, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: The Innate Immunity Adaptor SARM Translocates to the Nucleus to Stabilize Lamins and Prevent DNA Fragmentation in Response to Pro-Apoptotic Signaling

doi: 10.1371/journal.pone.0070994

Figure Lengend Snippet: A. Immunoblot analysis of HEK 293 whole cell lysates. Cells were transiently transfected with the designated expression vectors. B. HEK 293 cells were transiently transfected with both the SARM-eGFP vector and a two-fold molar excess of either empty Neo vector or the expression vector for the non-fluorescent NH 2 -terminal ARM-repeat region (ARM-Neo) or the non-fluorescent COOH-terminal TIR domain (TIR-Neo). Fixed cells were counterstained with propidium iodide (PI) to indicate nuclei. White scale bars = 10 µm. Results shown are representative of three independent experiments.

Article Snippet: Immunoblotting of nuclear extracts was performed using a polyclonal anti-SARM antibody (United States Biological) recognizing the C-terminus of SARM and a polyclonal antibody to lamin A/C (Abcam) that recognizes both full-length lamin A and C (∼70 kDa) and a 28 kDa lamin cleavage fragment.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation