mouse  (R&D Systems)

Journal: Autophagy

Article Title: Impaired lipophagy induced-microglial lipid droplets accumulation contributes to the buildup of TREM1 in diabetes-associated cognitive impairment.

doi: 10.1080/15548627.2023.2213984

Figure Lengend Snippet: Figure 4. Increased protein level of TREM1 in hyperglycemic plasma or HG-cultured microglia and in the hippocampal microglia of db/db mice. (A) the mRNA expression levels of TREM and TREML receptors were analyzed by RT-Qpcr and TREM1 protein expressions was detected via western blotting (B). ACTB was used as control. (C) Western blotting analysis of TREM1 protein expressions and (D) confocal images of TREM1 in BV2 cells treated with plasma of db/db mice. Scale bar: 50 μm. (E) Confocal images of TREM1 in HMC3 cells treated with T2DM patients plasma. The number of TREM1+ cells were quantified. Scale bar: 50 μm. (F) Representative Imaris 3D reconstructions of TREM1-positive expression in IBA+ microglia of the hippocampi of db/db and HFD/STZ mice. TREM1-positive cells were quantified. Scale bar: 50 μm. *P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Immunofluorescence and BODIPY staining of brain and cell sections Brain and cell sections were used for immunofluorescent staining with the following primary antibodies were used: PLIN2 (1:100; Fitzgerald LLC, 20 R-AP002), AIF1 (1:200; wako, 019– 19741), LC3B (1:100; ABclonal, A19665), SQSTM1 (1:100; Cell Signaling Technology [CST], 5114S), TREM1 (1:200; R&D Systems, MAB1187–100), RBFOX3 (1:200; Abcam, ab177487) GFAP (1:200; Genetex, GTX85454), MAP2 (1:100; CST, 4542S), and DLG4 (1:100; Abcam, ab238135).

Techniques: Clinical Proteomics, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Autophagy

Article Title: Impaired lipophagy induced-microglial lipid droplets accumulation contributes to the buildup of TREM1 in diabetes-associated cognitive impairment.

doi: 10.1080/15548627.2023.2213984

Figure Lengend Snippet: Figure 5. HG induced colocalization of TREM1 with LDs in microglial cells in vivo and in vitro. Representative immunostaining of TREM1 and PLIN2 in HMC3 cells cultured with plasma from T2DM patients (A) and HG (B). The number of TREM1+ cells in PLIN2− and PLIN2+ cells were quantified. Scale bar: 50 μm. (C and D) Representative micrographs of TREM1 and PLIN2 in different morphologies in HMC3 cells cultured with plasma from T2DM patients (C) and BV2 cells cultured with plasma from db/db mice (D). Scale bar: 10 μm. (E) Representative Imaris 3D reconstructions of colocalization of TREM1 and PLIN2 in the hippocampal microglia of db/ db mice (upper panel) and HFD/STZ mice (lower panel). The number of TREM1+ cells in PLIN2− and PLIN2+ cells were quantified. (n = 5 mice per group, scale bar: 15 μm). (F) Co-IP validation of TREM1 and PLIN2 interaction in HMC3 cells cultured with HG. (G) Western blotting analysis of the expression of TREM1 and its downstream protein, phospho-SYK in HG and RAPA treated HMC3. ACTB was used as control. *P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Immunofluorescence and BODIPY staining of brain and cell sections Brain and cell sections were used for immunofluorescent staining with the following primary antibodies were used: PLIN2 (1:100; Fitzgerald LLC, 20 R-AP002), AIF1 (1:200; wako, 019– 19741), LC3B (1:100; ABclonal, A19665), SQSTM1 (1:100; Cell Signaling Technology [CST], 5114S), TREM1 (1:200; R&D Systems, MAB1187–100), RBFOX3 (1:200; Abcam, ab177487) GFAP (1:200; Genetex, GTX85454), MAP2 (1:100; CST, 4542S), and DLG4 (1:100; Abcam, ab238135).

Techniques: In Vivo, In Vitro, Immunostaining, Cell Culture, Clinical Proteomics, Co-Immunoprecipitation Assay, Biomarker Discovery, Western Blot, Expressing, Control

Journal: Autophagy

Article Title: Impaired lipophagy induced-microglial lipid droplets accumulation contributes to the buildup of TREM1 in diabetes-associated cognitive impairment.

doi: 10.1080/15548627.2023.2213984

Figure Lengend Snippet: Figure 6. Suppression of TREM1 reversed impairment of lipophagy and accumulation of LDs in HG-stimulated microglia. (A) Schematic diagram for HG-impaired lipophagy, accumulation of LDs and TREM1 in microglia. (B) Western blotting analysis of TREM1, phospho-SYK, LC3B-I/II, SQSTM1 and PLIN2 expressions in Trem1- specific shRNA-infected BV2 cells. *P < 0.05, ** P < 0.01, *** P < 0.001. (C) Representative micrographs of BV2 cells co-treated with glucose and LP17. Lipophagy was visualized using SQSTM1 puncta, and LDs were visualized using PLIN2 puncta. The numbers of SQSTM1 puncta and PLIN2 puncta were quantified. Scale bar: 50 μm. ** P < 0.01: 25 mM vs 25 mM + LP17. (D and E) Western blotting analysis of phospho-SYK, LC3B-I/II, SQSTM1 and PLIN2 expressions in BV2 cells (D) and HMC3 cells (E) co-treated with glucose and LP17. ACTB was used as control. *P < 0.05, ** P < 0.01, *** P < 0.001. (F) Representative micrographs of lipophagy in HMC3 cells co- treated with glucose and LP17 via immunostaining with SQSTM1 and PLIN2. The number of SQSTM1 puncta and PLIN2 puncta were quantified. Scale bar: 50 μm. *P < 0.05, ** P < 0.01: 25 mM vs 25 mM + LP17.

Article Snippet: Immunofluorescence and BODIPY staining of brain and cell sections Brain and cell sections were used for immunofluorescent staining with the following primary antibodies were used: PLIN2 (1:100; Fitzgerald LLC, 20 R-AP002), AIF1 (1:200; wako, 019– 19741), LC3B (1:100; ABclonal, A19665), SQSTM1 (1:100; Cell Signaling Technology [CST], 5114S), TREM1 (1:200; R&D Systems, MAB1187–100), RBFOX3 (1:200; Abcam, ab177487) GFAP (1:200; Genetex, GTX85454), MAP2 (1:100; CST, 4542S), and DLG4 (1:100; Abcam, ab238135).

Techniques: Western Blot, shRNA, Infection, Control, Immunostaining

Journal: Autophagy

Article Title: Impaired lipophagy induced-microglial lipid droplets accumulation contributes to the buildup of TREM1 in diabetes-associated cognitive impairment.

doi: 10.1080/15548627.2023.2213984

Figure Lengend Snippet: Figure 7. Pharmacological blockade of TREM1 in db/db model improved cognitive function, reduced hippocampal neuronal degeneration and inhibited microglial inflammation. db/m and db/db mice were administrated with or without LP17 injection. (A) Platform crossing times, time spent in the target quadrant (P, Platform) and (B) average swimming speed of the MWM test (n = 10 mice for db/m, n = 8 mice for db/db). (C) Representative Imaris reconstructions of DLG4 in hippocampus and quantification of DLG4 puncta in the hippocampus of mice from different groups. (D) mRNA expression of inflammatory factors in the hippocampus of mice from different groups. *P < 0.05: db/m vs. db/db; #P < 0.05: db/db vs. db/db + LP17. (E) Representative micrographs of PLIN2 (upper panel, scale bar: 50 μm) and representative Imaris 3D reconstructions of colocalization of TREM1 (middle panel, scale bar: 15 μm) or SQSTM1 (lower panel, scale bar: 15 μm) with AIF1 in the hippocampus of mice from different groups. The numbers of PLIN2+AIF1+, TREM1+AIF1+ and SQSTM1 puncta per AIF1+ cells were quantified. *P < 0.05, **P < 0.01, ***P < 0.001. (F) Western blotting analysis of PLIN2 expressions in the hippocampus of db/m and db/db mice with or without LP17. **P < 0.01. (G) Proposed series of events involved in HG-impaired microglia and the blockade of TREM1 pathway with LP17 administration as a new strategy for DACI therapy.

Article Snippet: Immunofluorescence and BODIPY staining of brain and cell sections Brain and cell sections were used for immunofluorescent staining with the following primary antibodies were used: PLIN2 (1:100; Fitzgerald LLC, 20 R-AP002), AIF1 (1:200; wako, 019– 19741), LC3B (1:100; ABclonal, A19665), SQSTM1 (1:100; Cell Signaling Technology [CST], 5114S), TREM1 (1:200; R&D Systems, MAB1187–100), RBFOX3 (1:200; Abcam, ab177487) GFAP (1:200; Genetex, GTX85454), MAP2 (1:100; CST, 4542S), and DLG4 (1:100; Abcam, ab238135).

Techniques: Injection, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Endothelial TREM ‐1 receptor regulates the blood–brain barrier integrity after intracerebral hemorrhage in mice via SYK /β‐catenin signaling

doi: 10.1111/cns.14255

Figure Lengend Snippet: Inhibition of TREM‐1 reduced BBB leakage 72 h after ICH. (A) Pictures of brain slices 2 mm thickness with extravasated EB dyes in different groups. (B) Statistical analysis of EB extravasation. The error bars represent the mean ± SD, * p < 0.05 versus sham, # p < 0.05 versus ICH + Control peptide. N = 6 per group. (C) Immunohistochemistry for perivascular IgG staining surrounding hematoma.

Article Snippet: Anti‐mouse TREM‐1 rat IgG2a (0.25 μg/g; Thermo Fisher Scientific), as a specific TREM‐1 agonist, or control rat IgG2a (Thermo Fisher Scientific), was administered intracerebroventricularly.

Techniques: Inhibition, Control, Immunohistochemistry, Staining