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 membranes were blocked in PBS with 5% non-fat milk for 1 h and then incubated with their respective primary antibodies against rat anti-mouse CD11b (1:800, AbD Serotec, Oxford, UK), mouse anti-mouse CD86 (1:800, Abcam, Cambridge, MA, USA), and rat anti-mouse TREM2 (1:500, Merck Millipore, Temecula, CA, USA), and with antibodies purchased from Santa-Cruz Biotechnology (Santa Cruz, USA) that recognize mouse anti-mouse CD206 (1:100) and mouse anti-mouse Arg1 (1:100), and from Cell Signaling Technology (Danvers, MA, USA) that recognize rabbit anti-mouse phosphor-PI3K p85 Tyr458 (p-PI3K, 1:800), rabbit anti-mouse phospho-Akt Ser473 (p-Akt, 1:1,000).

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 membranes were blocked in PBS with 5% non-fat milk for 1 h and then incubated with their respective primary antibodies against rat anti-mouse CD11b (1:800, AbD Serotec, Oxford, UK), mouse anti-mouse CD86 (1:800, Abcam, Cambridge, MA, USA), and rat anti-mouse TREM2 (1:500, Merck Millipore, Temecula, CA, USA), and with antibodies purchased from Santa-Cruz Biotechnology (Santa Cruz, USA) that recognize mouse anti-mouse CD206 (1:100) and mouse anti-mouse Arg1 (1:100), and from Cell Signaling Technology (Danvers, MA, USA) that recognize rabbit anti-mouse phosphor-PI3K p85 Tyr458 (p-PI3K, 1:800), rabbit anti-mouse phospho-Akt Ser473 (p-Akt, 1:1,000).

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 membranes were blocked in PBS with 5% non-fat milk for 1 h and then incubated with their respective primary antibodies against rat anti-mouse CD11b (1:800, AbD Serotec, Oxford, UK), mouse anti-mouse CD86 (1:800, Abcam, Cambridge, MA, USA), and rat anti-mouse TREM2 (1:500, Merck Millipore, Temecula, CA, USA), and with antibodies purchased from Santa-Cruz Biotechnology (Santa Cruz, USA) that recognize mouse anti-mouse CD206 (1:100) and mouse anti-mouse Arg1 (1:100), and from Cell Signaling Technology (Danvers, MA, USA) that recognize rabbit anti-mouse phosphor-PI3K p85 Tyr458 (p-PI3K, 1:800), rabbit anti-mouse phospho-Akt Ser473 (p-Akt, 1:1,000).

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