Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: Characterization of TREM2 expression in different groups of BV2 cells. BV2 cells were transfected with control pC1 or pTREM2 to generate stable BV2/NC and BV2/TREM2 cells. Simultaneously, BV2 cells transfected with control or TREM2‐specific siRNAs for 48 h. The relative levels of TREM2 expression in BC2/NC and BV2/TREM2 were determined by quantitative RT‐PCR and Western blot. The relative levels of TREM2 to GADPH protein expression in the control and TREM2‐specific siRNA‐transfected cells were determined by Western blot. Data are representative images and expressed as the mean ± SD of each group of cells from three separate experiments. (A) Quantitative RT‐PCR analysis of TREM2 mRNA transcripts. (B) Western blot analysis of TREM2 expression in BV2/NC and BV2/TREM2 cells. (C) Quantitative RT‐PCR analysis of TREM2‐specific siRNA mRNA transcripts. (D) Western blot analysis of TREM2 expression in BV2/NC and BV2/ TREM2‐specific siRNAs cells. ** P < 0.01 versus the BV2 cells; ## P < 0.01 versus the BV2/NC; ^^ P < 0.01 versus the BV2 siRNA ctrl cells.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot

Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: Altered TREM2 expression modulates the LPS‐induced PI3K‐dependent cell proliferation in BV2 cells. BV2, BV2/NC, BV2/TREM2, BV2/siRNA, and BV2/siRNA‐TREM2 cells were pre‐treated in triplicate with, or without, LY294002, a PI3 K inhibitor, for 1 h and stimulated with, or without, LPS for 24 h. The cell viability of individual groups of cells were determined by the CCK8 assays. Data are expressed as the mean ± SD of each group of cells from three separate experiments. (A) Induction of TREM2 over‐expression mitigates LPS‐induced PI3K‐dependent cytotoxicity against in BV2 cells. (B) Knockdown of TREM2 expression enhances LPS‐induced PI3K‐dependent cytotoxicity against BV2 cells. ** P < 0.01 versus the BV2 cells; ## P < 0.01 versus the BV2/NC; ^^ P < 0.01 versus the BV2/NC or BV2/NC siRNA ctrl cells; && P < 0.01 versus the BV2/NC PI3K‐inhibitor or BV2/NC siRNA ctrl PI3K‐inhibitor cells.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Expressing, Over Expression

Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: Altered TREM2 expression changes LPS‐stimulated PI3K‐inhibited phagocytosis in BV2 cells. The different groups of cells were pre‐treated with, or without, the PI3K inhibitor and stimulated with, or without, LPS for 24 h. Subsequently, the different groups of cells were treated with fluorescence labeled latex beads at 37°C, 5% CO 2 for 1 h. After being washed, the cells were stained with rabbit anti‐mouse Iba‐1 and then Alexa Fluor 488‐goat anti‐rabbit IgG, followed by counterstained with DAPI. The cells were photoimaged and the percentages of phagocytosed (red color) cells in total DAPI+ cells were calculated. Data are representative images or expressed as the mean ± SD of each group of cells from three separate experiments. (A and B) Induction of TREM2 overexpression enhances LPS‐induced PI3K‐inhibited phagocytosis in BV2 cells. (C and D) Knockdown of TREM2 expression reduces LPS‐induced PI3K‐inhibited phagocytosis in BV2 cells. In Figure B: a. BV2 b. BV2+LPS c. BV2+TREM2 WT+LPS d. BV2+ TREM2 overexpression + LPS e. BV2+ TREM2 WT+ PI3K‐inhibitor + LPS, f: BV2+ TREM2 overexpression + PI3K‐inhibitor + LPS. Page 25 of 31 Cell Biology International For Review Only In Figure D, a. BV2 b. BV2+LPS c. BV2+SiRNA ctrl+LPS d. BV2+ SiRNA TREM2 + LPS e. BV2+ SiRNA ctrl + PI3K‐inhibitor + LPS f. BV2+ SiRNA TREM2 + PI3K‐inhibitor + LPS. ** P < 0.01 versus the BV2 cells. ## P < 0.01 versus the BV2/NC ^^ P < 0.01 versus BV2/NC or BV2/NC siRNA ctrl cells && P < 0.01 versus the BV2/NC PI3K‐inhibitor or BV2/NC siRNA ctrl PI3K‐inhibitor cells.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Expressing, Fluorescence, Labeling, Staining, Over Expression

Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: TREM2 reduces LPS‐induced PI3K‐enhanced apoptosis of BV2 cells. BV2, BV2/NC, BV2/TREM2, BV2/siRNA, and BV2/siRNA‐TREM2 cells were pre‐treated in triplicate with, or without, LY294002, a PI3K inhibitor, for 1 h and stimulated with, or without, LPS for 24 h. The percentages of apoptotic cells were determined by flow cytometry. Data are representative flow cytometry charts or expressed as the mean ± SD of each group of cells from three separate experiments. (A and B) Induction of TREM2 overexpression reduces LPS‐induced PI3K‐enhanced apoptosis of BV2 cells. (C and D) Knockdown of TREM2 expression increases LPS‐induced PI3K‐enhancedapoptosis of BV2 cells. ** P < 0.01 versus the BV2 cells ## P < 0.01 versus the BV2/NC ^^ P < 0.01 versus the BV2/NC or BV2/NC siRNA ctrl cells && P < 0.01 versus the BV2/NC PI3K‐inhibitor or BV2/NC siRNA ctrl PI3K‐inhibitor cells.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Flow Cytometry, Over Expression, Expressing

Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: TREM2 inhibits LPS‐stimulated PI3K‐dependent NO production and LDH activity in BV2 cells. BV2, BV2/NC, BV2/TREM2, BV2/siRNA, and BV2/siRNA‐TREM2 cells were pre‐treated in triplicate with, or without, LY294002, a PI3K inhibitor, for 1 h and stimulatedwith, or without, LPS for 24 h. The concentrations of NO in the supernatants of cultured cells were determined and the LDH activity in different groups of cells was analyzed. Data are expressed as the mean ± SD of each group of cells from three separate experiments. (A and B) Induction of TREM2 overexpression reduces LPS‐induced PI3K‐enhanced NO production and LDH activity in BV2 cells. (C and D) Knockdown of TREM2 expression increases LPS‐induced PI3K‐enhanced NO production and LDH activity in BV2 cells. ** P < 0.01 versus the BV2 cells ## P < 0.01 versus the BV2/NC ^^ P < 0.01 versus the BV2/NC or BV2/NC siRNA ctrl cells && P < 0.01 versus the BV2/NC PI3K‐inhibitor or BV2/NC siRNA ctrl PI3K‐inhibitor cells.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Activity Assay, Cell Culture, Over Expression, Expressing

Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: TREM2 modulates LPS‐stimulated cytokine production in BV2 cells. BV2, BV2/NC, BV2/TREM2, BV2/siRNA, and BV2/siRNA‐TREM2 cells were pre‐treated with, or without, LY294002, a PI3K inhibitor, for 1 h and stimulated with, or without, LPS for 24 h. The concentrations of TNF‐α, IL‐1β, IL‐10, and TGF‐β1 in the supernatants of different groups of cells were determined by ELISA. Data are expressed as the mean ± SD of each group of cells from three separate experiments. (A) Induction of TREM2 overexpression reduces LPS‐induced PI3K‐enhanced PI3K‐dependent TNF‐α, IL‐1β, but promotes the PI3K‐dpendent IL‐10 and TGF‐β1 expression in BV2 cells. (B) Knockdown of TREM2 expression enhances LPS‐induced PI3K‐dependent TNF‐α, IL‐1β, but decreases the PI3K‐dpendent IL‐10 and TGF‐β1 expression in BV2 cells. ** P < 0.01 versus the BV2 cells ## P < 0.01 versus the BV2/NC ^^ P < 0.01 versus the BV2/NC or BV2/NC siRNA ctrl cells && P < 0.01 versus the BV2/NC PI3K‐inhibitor or BV2/NC siRNA ctrl PI3K‐inhibitor cells.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Over Expression, Expressing

Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: LPS‐induced NF‐κB activation in BV2 cells. BV2 cells were stimulated with, or without LPS for 24 h. The relative levels of AKT and NF‐kB expression and phosphorylation to the control β‐actin in the different groups of cells were determined by Western blot assays. Data are representative images or expressed as the mean ± SD of each group of cells from three separate experiments. In Figure B: 1. BV‐2+TREM2‐WT+LPS 2. BV2+T REM2‐overexpression+LPS 3. BV2 +TREM2‐WT+ PI3K‐inhibitor + LPS 4. BV2 + TREM2‐overexpression + PI3K‐inhibitor + LPS. In Figure C: 1. BV2+siRNA‐ctrl+LPS 2. BV‐2+ siRNA‐TREM2+LPS 3. BV2 + siRNA‐ctrl +PI3K‐inhibitor + LPS 4. BV2 + siRNA‐TREM2 + PI3K‐inhibitor + LPS.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Activation Assay, Expressing, Western Blot, Over Expression

Journal: Cell Biology International

Article Title: TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF‐κB signaling

doi: 10.1002/cbin.10975

Figure Lengend Snippet: TREM2 prevents from LPS‐induced NF‐κB and PI3K activation in BV2 cells. BV2, BV2/NC, BV2/TREM2, BV2/siRNA, and BV2/siRNA‐TREM2 cells were pre‐treated with, or without, LY294002, a PI3K inhibitor, for 1 h and stimulated with, or without, LPS for 24 h. (A) Induction of TREM2 overexpression reduces the LPS‐induced PI3K and AKT activation in BV2 cells. (B) Knockdown of TREM2 expression enhances the LPS‐induced AKT and NF‐kB activation in BV2 cells. ** P < 0.01 versus the BV2 cells ## P < 0.01 versus the BV2/NC ^^ P < 0.01 versus the BV2/NC or BV2/NC siRNA ctrl cells && P < 0.01 versus the BV2/NC PI3K‐inhibitor or BV2/NC siRNA ctrl PI3K‐inhibitor cells.

Article Snippet: After washing five times with TBS, the coverslips were blocked with Immunol Staining Blocking Buffer (Beyotime) for 30 min, and then incubated with anti‐TREM2(1:1000 dilution, Bioss, Boston, USA)at 4°C overnight.

Techniques: Activation Assay, Over Expression, Expressing