Journal: The Journal of Biological Chemistry

Article Title: Wild-type sTREM2 blocks Aβ aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aβ aggregation

doi: 10.1016/j.jbc.2021.100631

Figure Lengend Snippet: Aβ oligomers induce TREM2 proteolysis and sTREM2 release, which then binds Aβ oligomers, but R47H sTREM2 binds less . A , western blot of cell lysate and unprocessed supernatant (sTREM2) of HEK293 cells coexpressing human DAP12 and full-length N-terminally-tagged wild-type (WT) human TREM2 (FL-TREM2) 16 h after adding Aβ oligomers. This blot and those for Aβ monomers and fibrils are reproduced in Figure S5 for comparison. B , quantification of sTREM2 release from transfected HEK293 cells expressing wild-type ( green line ) and R47H TREM2 ( red line ). C , quantification of sTREM2 release from wild-type TREM2 expressing HEK293 cells induced by doses of Aβ oligomers ( red line ), monomers ( green line ), or fibrils ( blue line ). For both ( B and C ) error bars = SEM; ∗ p < 0.05 ∗∗ p < 0.01 ∗∗∗ p < 0.001, n = 3 independent experiments; one-way ANOVA with Tukey's post-hoc multiple comparisons test. D , example field of single-molecule TIRF imaging of mixture of Aβ oligomers ( green ) and wild-type TREM2 ectodomain ( red ), where colocalized spots appear yellow . Scale bar: 1 micron. Magnified image of three sections of field at right . E , proportion of monomeric or oligomeric Aβ colocalized with wild-type sTREM2. F , proportion of Aβ oligomers colocalized with wild-type or R47H TREM2 ectodomain. For ( E and F ), error bars = SEM; ∗∗∗∗ p < 0.0001, n = 3 independent preparations, each analyzed in nine fields each; two-tailed t -test of significance.

Article Snippet: Function-blocking TREM2 antibody (Clone 78.18, 25 μg/ml for blocking assay, AbD Serotec catalogue number MCA4772EL) and a control antibody (Rat IgG1, AbD Serotec) were used for blocking experiments.

Techniques: Western Blot, Transfection, Expressing, Imaging, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Wild-type sTREM2 blocks Aβ aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aβ aggregation

doi: 10.1016/j.jbc.2021.100631

Figure Lengend Snippet:

Article Snippet: Function-blocking TREM2 antibody (Clone 78.18, 25 μg/ml for blocking assay, AbD Serotec catalogue number MCA4772EL) and a control antibody (Rat IgG1, AbD Serotec) were used for blocking experiments.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: TREM2 Regulates Heat Acclimation-Induced Microglial M2 Polarization Involving the PI3K-Akt Pathway Following EMF Exposure

doi: 10.3389/fncel.2019.00591

Figure Lengend Snippet: Effects of HA on the expression of triggering receptor expressed on myeloid cells-2 (TREM2) and the phosphorylation of PI3K and Akt in EMF-stimulated N9 cells. N9 cells were pretreated with or without a 72-h HA process (20/4-h cycle of 37°C/39.5°C) and then exposed to 2.45-GHz EMF or sham-exposed for 20 min. Levels of TREM2 (A) , and phosphorylation of PI3K and Akt (B) in total cell lysates were analyzed using Western blotting, and the corresponding densitometric analyses were represented. Data are presented as means ± SEM of three independent experiments. * P < 0.05 vs. the sham-exposed control group; # P < 0.05 vs. the EMF-exposed group.

Article Snippet: The cells were incubated with rat anti-mouse TREM2 (Merck Millipore), rabbit anti-mouse phosphor-Ser473-Akt (Cell Signaling Technology, Danvers, MA, USA), and/or mouse anti-mouse CD206 (Santa Cruz) antibodies at 37°C for 1 h. After being washed, the slides were incubated for 1 h at 37 °C with rabbit anti-mouse AlexaFluor 488 (Life Technologies, Carlsbad, CA, USA), goat anti-rat CF568 (Sigma–Aldrich, St. Louis, MO, USA), and chicken anti-rabbit CF633 antibodies (Sigma–Aldrich, St. Louis, MO, USA) in the dark.

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: TREM2 Regulates Heat Acclimation-Induced Microglial M2 Polarization Involving the PI3K-Akt Pathway Following EMF Exposure

doi: 10.3389/fncel.2019.00591

Figure Lengend Snippet: Effects of TREM2 esiRNA on M2 microglial phenotype regulation in EMF-stimulated N9 cells with HA preconditioning. N9 cells were transfected with or without TREM2 esiRNA (20 nM) at 60 h during the uncompleted 72-h HA and then continuously cultured 12 h to complete the HA process and the 24 h transfection of TREM2 esiRNA. Western blotting quantification of TREM2 (A) , and M2 markers CD206 and Arg1 (C) , and ELISA of anti-inflammatory cytokines IL-4 and IL-10 (B) production of in either control or HA-plus-EMF-treated N9 cells with or without TREM2 esiRNA. Data are presented as means ± SEM of three independent experiments. * P < 0.05 vs. the non-HA and sham-exposed control group; # P < 0.05 vs. the HA-plus-EMF-exposed group. (D) Confocal immunofluorescence microscopy was performed on cultures that were immunoreacted with antibodies against TREM2 and CD206 with esi-TREM2 treatment in HA-plus-EMF-treated N9 cells. Scale bar: 20 μm.

Article Snippet: The cells were incubated with rat anti-mouse TREM2 (Merck Millipore), rabbit anti-mouse phosphor-Ser473-Akt (Cell Signaling Technology, Danvers, MA, USA), and/or mouse anti-mouse CD206 (Santa Cruz) antibodies at 37°C for 1 h. After being washed, the slides were incubated for 1 h at 37 °C with rabbit anti-mouse AlexaFluor 488 (Life Technologies, Carlsbad, CA, USA), goat anti-rat CF568 (Sigma–Aldrich, St. Louis, MO, USA), and chicken anti-rabbit CF633 antibodies (Sigma–Aldrich, St. Louis, MO, USA) in the dark.

Techniques: esiRNA, Transfection, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Immunofluorescence, Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: TREM2 Regulates Heat Acclimation-Induced Microglial M2 Polarization Involving the PI3K-Akt Pathway Following EMF Exposure

doi: 10.3389/fncel.2019.00591

Figure Lengend Snippet: Effects of TREM2 esiRNA and the PI3K inhibitor LY294002 on the phosphorylation of PI3K and Akt in EMF-stimulated N9 cells with HA preconditioning. N9 cells were transfected with or without TREM2 esiRNA (20 nM) at 60 h during the uncompleted 72-h HA and then continuously cultured 12 h to complete the HA process and the 24 h transfection of TREM2 esiRNA. Alternatively, N9 cells were treated with or without LY294002 (10 μM) for 30 min prior to the end of the 72-h HA process. Levels of PI3K and Akt phosphorylation in total cell lysates were analyzed using Western blotting, and the corresponding densitometric analyses were represented. Data are presented as means ± SEM of three independent experiments. * P < 0.05 vs. the non-HA and sham-exposed control group; # P < 0.05 vs. the HA-plus-EMF-exposed group.

Article Snippet: The cells were incubated with rat anti-mouse TREM2 (Merck Millipore), rabbit anti-mouse phosphor-Ser473-Akt (Cell Signaling Technology, Danvers, MA, USA), and/or mouse anti-mouse CD206 (Santa Cruz) antibodies at 37°C for 1 h. After being washed, the slides were incubated for 1 h at 37 °C with rabbit anti-mouse AlexaFluor 488 (Life Technologies, Carlsbad, CA, USA), goat anti-rat CF568 (Sigma–Aldrich, St. Louis, MO, USA), and chicken anti-rabbit CF633 antibodies (Sigma–Aldrich, St. Louis, MO, USA) in the dark.

Techniques: esiRNA, Phospho-proteomics, Transfection, Cell Culture, Western Blot, Control