goat anti trem2 r d system af1828  (R&D Systems)

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: TREM2 agonism induces PLC activity in iPSC- derived macrophages. ( A ) Representative Western blot showing phosphorylation of SYK after TREM2 stimulation for 5 min using a TREM2-specific antibody (AF1828, R&D Systems) in Parent and TREM2 Ko cells. Full immunoblot images are presented in Supplementary Fig. 6. ( B ) Ca 2+ flux induced by TREM2 ligation in Parent cells is absent in TREM2 Ko cells. n = 4. ( C) SYK inhibitor BIIB-057 (SYKi) reduces TREM2 antibody-evoked Ca 2+ signal in a dose-dependent manner in Parent cells. n = 3. ( D) TREM2-induced IP1 accumulation determined by HTRF assay is prevented in TREM2 Ko cells. n = 3, data shown represent mean ± SEM, ( B , D ) two-way ANOVA followed by Bonferroni’s multiple comparison test, ( C ) one-way ANOVA followed by Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Activity Assay, Derivative Assay, Western Blot, Phospho-proteomics, Ligation, HTRF Assay, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: PLCγ2 Ko leads to dysregulation of cell surface marker expression and morphological changes in iPSC-derived macrophages. ( A ) Western blot confirms lack of PLCγ2 protein in PLCγ2 Ko and shows reduction in TREM2 expression in PLCγ2 KO macrophages compared to Parent. n = 4. Full immunoblot images are presented in Supplementary Fig. 7. ( B ) Levels of soluble TREM2 detected in cell supernatant 8 days after plating are lower in PLCγ2 Ko cells compared to Parent. n = 4, ( C ) Macrophage surface markers CD11b, CD14, and CD45 were measured by flow cytometry, compared to relevant isotype IgG. Annotations indicate frequency of marker positivity in the Parent line. ( D) Morphology of macrophage lines was determined by phalloidin staining and analysis of cell roundness and cell area. Scale bar 50 μm. n = 4, data shown represent mean ± SEM, One-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Marker, Expressing, Derivative Assay, Western Blot, Flow Cytometry, Staining, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: PLCγ2 regulates TREM2-mediated signalling in iPSC- derived macrophages. ( A ) Representative Western blots showing phospho-SYK after TREM2 ligation using AF1828 in Parent and PLCγ2 Ko cells. ( B ) Quantification of SYK phosphorylation after TREM2 ligation shows no effect of PLCγ2 deficiency, n = 4. Full immunoblot images are presented in Supplementary Fig. 9. ( C ) pSYK HTRF assay confirms similar phosphorylation levels upstream of PLCγ2 upon TREM2 stimulation in Parent and PLCγ2 Ko cells, n = 3. ( D ) Ca 2+ flux induced by TREM2 ligation is prevented in PLCγ2 Ko cells. n = 3 ( E ) PLCγ2 deficiency abolishes TREM2-induced IP1 production as determined by HTRF assay. n = 3 ( F ) Representative Western blots and ( G ) quantification of ERK1/2 phosphorylation shows no major difference between Parent and PLCγ2 Ko cells. Full immunoblot images are presented in Supplementary Fig. 10. Data shown represent mean ± SEM, two-way ANOVA followed by Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Derivative Assay, Western Blot, Ligation, Phospho-proteomics, HTRF Assay, Comparison

Journal: Scientific Reports

Article Title: PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages

doi: 10.1038/s41598-021-96144-7

Figure Lengend Snippet: Primary antibodies used for Western blot.

Article Snippet: A 384-well source plate containing 5 × concentrated stimuli ATP (final concentration 0.5 mM, Sigma) or TREM2 antibody AF1828 (final concentration 1, 2, 5 or 10 μg/mL, R&D Systems) was prepared for transfer onto the macrophages.

Techniques: Western Blot

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Antibodies

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Reduced cell surface localization of R47H TREM2 does not impair antibody-mediated activation of TREM2 in pMac. a Schematic of microglia phenotypes investigated in this study. b , c Reduced cell surface expression of R47H TREM2. Cell surface proteins on pMac were biotinylated and pulled down, the level of TREM2 protein enrichment was measured by Western blotting vs whole cell lysate, probed on separate blots. c Means ± SEM, for N = 5 harvests measured on separate Western blots. 2-tailed paired t-test: ** p = 0.0011 for R47H versus WT. d Increased sTREM2 production from R47H TREM2 pMac. sTREM2 were measured from unstimulated pMac supernatants by ELISA. Means ± SEM, for N = 3 harvests measured on same ELISA plate, data for each harvest was normalised to the average cell count. 1-way ANOVA, with Dunnett’s post hoc test: p = 0.0005 R47H versus WT (***), p = 0.0066 KO versus WT (**). e , f Co-localization of TREM2 with subcellular compartment markers in fixed and permeabilized pMac, images are a confocal slice at 4 μm, taken with Opera Phenix microscope. Inset panels are × 3 magnification of a selected cell. Calnexin used as a marker for ER, TGN46 for TGN, and LAMP1 for lysosomes. f Co-localization expressed as a ratio of TREM2 intensity to compartment marker intensity, in regions automatically segmented by high marker staining. Means ± SEM, for N = 3 harvests, each in triplicate wells. 2-way ANOVA, with Bonferroni’s post hoc test, no significant differences. g , h TREM2-activating antibody stimulation (used at concentration of 2.4 μg/1 × 10 6 cells and 3.84 μg/mL, for 10 min) of both WT and R47H TREM2 pMac caused SYK phosphorylation, measured by Western blotting. No response seen in TREM2 antibody-stimulated TREM2 KO cells, or cells treated with a goat IgG isotype control. h Means ± SEM, for N = 3 harvests measured on separate Western blots. 2-way ANOVA, with Sidak’s post hoc test, pairwise comparisons to WT for each treatment: p < 0.001 for KO stimulated with TREM2 Ab versus WT (****). i Calcium response in response to TREM2 antibody is similar in WT and R47H TREM2 pMac, measured by peak Fluo4-AM fluorescence, normalised to minimum fluorescence and cell number. Means ± SEM, for N = 3 harvests. 2-way ANOVA, with Dunnett’s multiple comparison test, pairwise comparisons to WT for each treatment: p = 0.0049 for KO stimulated with TREM2 Ab versus WT (**)

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Activation Assay, Expressing, Protein Enrichment, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Microscopy, Marker, Staining, Concentration Assay, Control, Fluorescence, Comparison

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Phagocytosis of dead SH-SY5Ys and synaptosomes is reduced in TREM2 KO, but not R47H TREM2, relative to WT pMac. a After 3 h of phagocytosis of pHrodo-labelled dead SH-SY5Ys, immunofluorescence staining shows that TREM2 is highly recruited to the phagocytic cup (marked by white arrow) during engulfment of cells expressing the neuronal marker TUJ1, whereas in b TREM2 is lost before maturation to RAB9+ endosomes (marked by white arrow). c – f Phagocytosis is impaired in TREM2 KO pMac only. Representative images of phagocytosis of SH-SY5Ys ( c ) or synaptosomes ( e ) shown in yellow, by pMac (red cytoplasm and blue nucleus), taken at 3 h with INCell 6000. Inset is a section of the image magnified 3-fold. d , f Means were quantified for the parameters: number of spots per cell, sum of spot areas (μm 2 ) per cell, percentage of cells containing phagocytosed particles per field. Data was normalised to mean for each genotype per experiment. Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, Dunnett’s post hoc test, pairwise comparisons to the WT for each time: * p < 0.05, ** p < 0.01, *** p < 0.001. g , h Phagocytosis of dead SH-SY5Ys results in SYK phosphorylation, which is unaffected in R47H TREM2 cells but attenuated in TREM2 KO line, measured by Western blotting at 0.5, 1, and 2 h after phagocytosis initiation. h Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to the WT for each time: p = 0.0008 at 0.5 h stimulation for KO versus WT (***)

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Immunofluorescence, Staining, Expressing, Marker, Western Blot

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Divergent phenotypes in TREM2 KO and R47H TREM2 pMac, regarding cell morphology, migration, survival, and inflammatory responses. a , b TREM2 KO pMac are smaller and rounder than WT. Cell morphology measured by microscopy of pMac stained with CellTracker Deep Red, cells were fixed and imaged on INCell 6000 microscope. Representative images shown ( a ), and mean cell area (μm 2 ) and roundness were automatically quantified from 9 fields per well in triplicate wells using Columbus software ( b ). Means ± SEM, for N = 3 harvests. 1-way ANOVA with Dunnett’s post hoc test: p = 0.019 for cell area in KO versus WT (*), p = 0.007 for roundness in KO versus WT (**). c , d TREM2 KO pMac migrate slower towards C5a, but not ADP, compared with WT. Cell migration measured by transwell assay. Migration of pMac towards 30 μM ADP or 3 nM C5a is compared to unstimulated migration over 6 h ( c ), and inhibitors used to unmask the contribution of purinergic receptors P2RY1 (3 μM MRS2179), P2RY12 (30 μM PSB0739), and P2RY13 (10 μM MRS2211) to ADP-induced migration ( d ). Data is expressed as the percentage of migrated cells and was normalised to average migration for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA with Dunnett’s post hoc test. Black annotations compare stimulation to unstimulated control, or ADP + purinergic inhibitors to the ADP-only control. Grey annotations compare R47H or KO versus WT for each stimulation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant. e , f TREM2 KO pMac exhibit increased cell death in the absence of M-CSF, whereas WT and R47H pMac remain viable. Survival after M-CSF withdrawal measured at 3, 7, and 10 days, with a comparison between M-CSF-deficient condition ( e ) to full-media controls ( f ) in the same plate. Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to WT for each time: M-CSF-deficient KO versus WT at 3 days p = 0.0051 (**), at 7 days p = 0.003 (**), and at 10 days p = 0.0001 (***). Full media KO versus WT at 3 days p = 0.035 (*). g , h Comparable secretion of TNF and IL-6 by pMac in response to E. coli LPS (100 ng/mL, 4 h) ± priming by interferon-γ (100 ng/mL, 24 h prior to LPS). Concentration of TNF ( g ) and IL-6 ( h ) was measured by separate ELISAs of the same supernatants, normalised to cell number, and normalised to the average pg/mL/cell for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to WT for each stimulation: p = 0.0097 for TNF secreted from R47H versus WT with LPS ± IFNγ stimulation (**). As expected, IFNγ-priming enhances TNF and IL-6 secretion upon LPS stimulation, significance is not depicted for clarity, but p < 0.001 for IFNγ+LPS versus LPS, for TNF and IL-6 of all genotypes

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Migration, Microscopy, Staining, Software, Transwell Assay, Control, Comparison, Concentration Assay

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Top DEGs of R47H TREM2 and TREM2 KO pMac overlap; furthermore, the R47H DEGs that are shared with TREM2 KO represent numerous biological processes, distributed between five protein-protein interaction (PPI) modules. a Heatmap of top upregulated and top downregulated DEGs for R47H TREM2 and TREM2 KO, with the relative expression for each gene represented by colours on the corresponding row. A selection of “relevant genes” was also included. Unbiased clustering (dendrogram on the left) shows that genes separate into two major clusters based on whether they are upregulated or downregulated in TREM2 KO or R47H TREM2, and there is no genotype-specific segregation. b Five modules of functionally related DEGs identified by PPI network analysis of TREM2 KO DEGs, with enriched gene ontology (GO) terms shown. R47H DEGs were only included where they were also differentially expressed in TREM2 KO and were overlaid onto clusters identified using TREM2 KO data. Clusters identified numerically on the x -axis, with TREM2 KO on the left and R47H TREM2 on the right, showing a similar pattern of dysregulated cell functions. Number of DEGs represented by circle size, and p value represented by colour

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Expressing, Selection

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Transcriptomics reveal high overlap in dysregulated genes of R47H TREM2 and TREM2 KO pMac, relative to WT. a Validation of pMac “microglial identity” by dendrogram comparison to Abud et al. transcriptomes of iPS cells, iPSC-haematopoietic progenitor cells (iHPC), iPSC-microglia-like cells (iMGL), iMGL without TGFβ1 or without CD200 supplementation, iMGL co-cultured with rat cortical neurons, blood-derived monocytes, blood-derived dendritic cells, and primary foetal and adult human microglia . b Plot of principal component analysis (PCA) with the first two principal components separating the RNA-seq samples by differentiation age and genotype. Ages represented by shapes, and genotypes represented by colour. c Venn diagram of differentially expressed genes (DEGs) identified relative to the WT line, showing the overlap between R47H TREM2 and TREM2 KO DEGs. DEGs are separately categorised as “upregulated” or “downregulated” relative to the WT

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Comparison, Cell Culture, Derivative Assay, RNA Sequencing Assay

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Extracellular matrix-adhesion modifiers and adhesion to vitronectin are dysregulated in R47H TREM2 and TREM2 KO pMac. TGFβ treatment does not rescue adhesion deficits. a Validation of selected RNA-seq hits by qRT-PCR of unstimulated pMac. Means ± SEM, for N = 7 harvests, including the 3 samples originally used for RNA-seq (open symbols) plus 4 samples harvested independently from a separate differentiation (filled symbols). Repeated-measures 1-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to the WT. b Effect of TGFβ-stimulation (50 ng/mL, 24 h) on mRNA levels of selected RNA-seq hits, measured by qRT-PCR. Means ± SEM, for N = 3 samples harvested independently to Fig. a. Two-way ANOVA, with Sidak’s post hoc test. c TREM2 KO pMac secrete reduced levels of TGFβ1 compared with WT, and TGFβ1 secretion is partly SYK-dependent. Total (inactive and active) TGFβ1 measured from supernatants by ELISA, cells treated ± OXSI-2 (2 μM, 24 h) to inhibit SYK. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. d Fibronectin protein expression is reduced in both R47H TREM2 and TREM2 KO versus WT. Fibronectin measured by Western blotting of pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Means ± SEM, quantified for N = 3 harvests on separate blots. Two-way ANOVA, with Sidak’s post hoc test. TGFβ1 vs unstimulated was not significant. e αVβ3 complex formation is reduced in both R47H TREM2 and TREM2 KO versus WT. Intact surface integrins αVβ3 and αVβ5 measured by flow cytometry in pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Data is expressed as the difference in median fluorescence intensity of the specific antibody versus isotype control, normalised (by subtraction) to the average for the harvest. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. f Adhesion to vitronectin is reduced for both R47H TREM2 and TREM2 KO versus WT, and treatment with TGFβ (50 ng/mL, 24 h prior to assay) increases αVβ3/5-dependent adhesion. Adhesion measured after 3 h by crystal violet colorimetric assay, and normalised to BSA-blocked wells (by division, and the result subtracted from 1). αVβ3/5 inhibitor (10 μM cilengitide) was added at the start of the assay to determine αVβ3/5-specific adhesion to vitronectin (striped bars). Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Dunnett’s post hoc test. Black annotations compare stimulations to unstimulated control. Grey annotations compare R47H or KO versus WT for each condition. WT = grey circles, R47H = orange squares, TREM2 KO = burgundy triangles. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Flow Cytometry, Fluorescence, Control, Colorimetric Assay

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Top 5 upstream regulators predicted to oppose TREM2 KO (group 1 genes)

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Activation Assay, Recombinant

Journal: Alzheimer's Research & Therapy

Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

doi: 10.1186/s13195-020-00709-z

Figure Lengend Snippet: Top 5 upstream regulators predicted to mimic TREM2 KO (group 1 genes)

Article Snippet: Stimulations with a TREM2-activating antibody (goat polyclonal antibody against human TREM2 AF1828 from R&D Systems) used a concentration of 2.4 μg/1 × 10 6 cells (3.84 μg/mL) for 10 min. Stimulations with dead SH-SY5Ys used a ratio of 3:1 SH-SY5Ys to pMac, with an initial 1 h incubation at 4 °C to allow cells to settle, followed by incubation at 37 °C for the time periods indicated.

Techniques: Activation Assay

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 1. TREM2 Binds to Ab (A and B) The binding of Ab1-42 monomers or oligomers with Fc, TREM2-Fc, or TREM1-Fc. Representative images (A) and quantified densi- tometric analysis (B). All values were normalized to input. M, marker; n = 5 independent experiments; ***p < 0.001 by unpaired t test. (C) Levels of biotin-oAb1-42 bound to immobilized Fc/Fc-fusion proteins were normalized to maximal Ab/TREM2-Fc interactions. n = 3 independent experiments. (D) Levels of Fc/Fc-fusion proteins bound to immobilized biotin-oAb1-42 were normalized to maximal TREM2-Fc/Ab binding. n = 3 indepen- dent experiments. (E and F) Bio-layer interferometry-binding kinetics of TREM2-His (E) or TREM1-His (F) incubated with immobilized biotin-oAb1-42 (curves correspond to His-tagged proteins with the following concen- trations: 222.2 nM [top] and 74.1, 24.7, 8.23, and 2.74 nM [bottom]; in E and F). (G) Maximal levels of biotin-oAb1-42 bound to im- mobilized wild-type (WT) TREM2-Fc and R47H and R62H mutants (normalized to WT). n = 3 in- dependent experiments; **p < 0.01, one-way ANOVA followed by Dunnett’s post hoc test. (H) Representative images of biotin-oAb1-42 (red) bound to BV2 cells overexpressing TREM2 or BV2 control (DAPI, blue; scale bar, 10 mm). n = 5 independent experiments; **p < 0.01, unpaired t test. (I and J) Levels of Ab1-42 co-precipitated with TREM2 in lysates from APP transgenic TgCRND8 mice (I) or human AD brains (J) were determined by ELISA. Bound Ab was normalized to the IgG control from each sample (n = 5 for TgCRND8, n = 6 for human AD). All values represent mean ± SD. See also Figure S1 and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Binding Assay, Marker, Incubation, Control, Transgenic Assay, Enzyme-linked Immunosorbent Assay

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 2. TREM2 Deficiency Modulates Microglia-Mediated Ab Degradation and Ab-Induced Depolarization (A–D) TREM2 deficiency attenuates microglia- mediated Ab degradation. Cultured microglia from WT or TREM2 KO mice were incubated with 200 nM FAM-Ab1-42 aggregates at 37C for the time indicated in (A), and they were washed/re- incubated in Ab-free media with or without MG132 (B–D) or chloroquine (CQ) (C and D). FAM-Ab in WT or TREM2 KO microglia at different time points was determined by flow cytometry (A and B) or confocal microscopy (C). All values were normal- ized to WT FAM-Ab levels 2 hr after uptake. Quantification of FAM-Ab fluorescence is shown in (D). Scale bar, 20 mm. n = 3 independent mea- surements; *p < 0.05, **p < 0.01, and ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test. (E) RMP in WT and TREM2 KO microglia with or without 6-hr exposure to oAb1-42 (5 mM). WT-control, n = 7; WT-Ab, n = 8; KO-control, n = 8; KO-Ab, n = 7; *p < 0.05, **p < 0.01, and ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test. (F) Representative inward currents obtained from primary microglia. (G) TREM2 KO microglia showed significantly reduced peak inward currents (n = 18 cells) when compared to WT (n = 9 cells). ***p < 0.001, unpaired t test. (H) At -150mV, Ab1-42 oligomers (10 min) reduce current amplitude in WT, but not in TREM2 KO microglia. All values were normalized to baseline for each cell. n = 5 cells for each group; *p < 0.05, paired t test. Electrophysiological recordings were taken from 4 independent microglial cultures. All values represent mean ± SD. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Cell Culture, Incubation, Cytometry, Confocal Microscopy, Control

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 3. TREM2 Deficiency Alters Microglia Cytokine Expression, Secretion, and Downstream Signaling in Response to Ab (A) IL-6, MIP-1a, or Arg1 expression was determined by qRT-PCR in WT or TREM2 KO microglia in the presence or absence of 1 mM Ab. n = 3 independent experiments; *p < 0.05 and **p < 0.01, two-way ANOVA followed by Tukey’s post hoc test. (B and C) Cytokine levels in conditioned media from WT or TREM2 KO microglial cultures treated with oAb1-42 for 24 (B) or 48 hr (C) were quantified by ELISA. All values were normalized to WT-Ab (0 mM) levels for each time point. n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Neuron

Article Title: TREM2 Is a Receptor for β-Amyloid that Mediates Microglial Function.

doi: 10.1016/j.neuron.2018.01.031

Figure Lengend Snippet: Figure 4. Microglial Migration, Ab Degrada- tion, and Downstream Effects on Microglial Proliferation and Apoptosis Are Altered in TREM2 KO Mice (A–C) Analysis of WT or TREM2 KO brain at 16 hr after FAM-Ab injection. n = 8 for WT, n = 9 for KO mice. (A) Representative images of Ab-injected regions. Scale bar, 20 mm. (B) Quantified average values for total Ab/IBA1- positive (IBA1+) cells. **p < 0.01, nonparametric t test. (C) Quantified Ab deposition area. (D and E) Ab staining analysis in WT or TREM2 KO mouse hippocampus 3 days after oAb1-42 injection. (D) Representative images (scale bar, 300 mm for 83 magnification; scale bar, 60 mm for 403 magnification). (E) Quantified values of the deposition area. n = 9 for WT, n = 8 for KO mice; *p < 0.05, nonparametric t test. (F and G) Immunostaining of PCNA (red) (F), cleaved-caspase-3 (C-caspase-3, red) (G), IBA1 microglial markers (green), and DAPI (blue) in the hippocampus of WT or TREM2 KO mice with con- trol vehicle or oAb injection as indicated. PCNA or c-caspase-3-positive microglial cells are indicated by white circles. Adjacent graphs represent quan- tified overlapping IBA1/PCNA- (F) or IBA1/C-cas- pase-3- (G) stained cells from 8–12 mice per group. Scale bar, 20 mm. *p < 0.05 and **p < 0.01, two-way ANOVA followed by Tukey’s post hoc test. (H) Reconstructed 3D microscopy images of IBA1-stained (white) microglia. Average process length per microglia was quantified and shown in the adjacent graph. n = 9–12 mice/group. Scale bar, 10 mm. ***p < 0.001, two-way ANOVA followed by Tukey’s post hoc test. All graphs represent mean ± SD. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-Ab1-12 (B436) (Huang et al., 2016) N/A Rabbit anti-human TREM2 Cell Signaling Technology Cat# 91068 RRID:AB_2721119 Goat anti-human TREM2 R&D Systems Cat# AF1828 RRID:AB_2208689 Sheep anti-mouse TREM2 R&D Systems Cat# AF1729 RRID:AB_354956) Mouse anti-Myc Thermo Fisher Scientific Cat# 13-2500 RRID:AB_2533008 Mouse anti-Gas6 Santa Cruz Biotechnology Cat# sc-376087 RRID:AB_10989223 Rabbit anti-phospho-SYK Thermo Fisher Scientific Cat# MA5-14918 RRID:AB_10989513 Rabbit anti-SYK Cell Signaling Technology Cat# 13198 RRID:AB_2687924 Rabbit anti-DAP12 Cell Signaling Technology Cat# 12492 RRID:AB_2721120 Mouse anti-b-actin Sigma Cat# A2228 RRID:AB_476697 Mouse anti-LAMP1 Santa Cruz Biotechnology Cat# sc-20011 RRID:AB_626853 Rabbit anti-phospho-GSK3b Cell Signaling Technology Cat# 5558 RRID:AB_10013750 Rabbit anti-GSK3b Cell Signaling Technology Cat# 12456 RRID:AB_2636978 Rabbit anti-PCNA Cell Signaling Technology Cat# 13110 RRID:AB_2636979 Rabbit anti-cleaved-caspase 3 Cell Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab5076 RRID:AB_2224402 Anti-Ab antibody MOAB-2 Biosensis Cat# M-1586-100 RRID:AB_2492497 Biological Samples For patient information, see Table S1 N/A Chemicals, Peptides, and Recombinant Proteins MG132 EMD Millipore Cat# 474790 Chloroquine Cell Signaling Technology Cat# 14774 Margatoxin Alomone Labs Cat# STM-325 Tertiapin Alomone Labs Cat# STT-250 Ab1-42 peptides Anaspec Cat# AS-20276 Biotin-Ab1-42 peptides Anaspec Cat# AS-23523 FAM-Ab1-42 peptides Anaspec Cat# AS-23525 Recombinant human IgG1 Fc R&D Systems Cat# 110-HG Recombinant human TREM2-Fc protein R&D Systems Cat# 1828-T2 Recombinant human TREM1-Fc protein R&D Systems Cat# 1278-TR Recombinant human CD36-Fc protein R&D Systems Cat# 1955-CD Recombinant human RAGE-Fc protein R&D Systems Cat# 1145-RG Recombinant human Gas6 protein R&D Systems Cat# 885-GSB Recombinant human MerTK-Fc protein R&D Systems Cat# 891-MR Recombinant human TREM2-His protein Sino Biological Cat# 11084-H08H Recombinant human TREM1-His protein Sino Biological Cat# 10511-H08H Critical Commercial Assays Human Ab1-42 ELISA kit Thermo Fisher Scientific Cat# KHB3441 Dynabeads protein G Thermo Fisher Scientific Cat# 10004D QuikChange II Site-Directed Mutagenesis kit Agilent Technologies Cat# 200523 Lipojet SignaGen Laboratories Cat# SL100468 (Continued on next page) e1 Neuron 97, 1023–1031.e1–e7, March 7, 2018

Techniques: Migration, Injection, Staining, Immunostaining, Microscopy