Figure 3. sTREM2 activates the Akt–GSK3β–β-catenin and the NF-κB signaling pathways in microglia. (A) Microglia obtained from WT or Trem2-KO mice were treated with 20 nM Fc or sTREM2-Fc protein for 24 h after GM-CSF withdrawal. The protein levels of phospho-Akt (p-Akt, Ser473), total-Akt (T-Akt), phospho-GSK3β (p-GSK3β, Ser9), total-GSK3β (T-GSK3β), and β-catenin were analyzed by Western blotting. (B) Quantification of the relative levels of p-Akt, p-GSK3β and β-catenin (n = 4 per group, unpaired Student’s t test). The protein level of Fc-treated WT microglia served as a control. (C) Microglia from WT mice were pretreated with 25 µM of a PI3K/Akt inhibitor (LY-294002) for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 24 h. The protein levels of p-Akt, T-Akt, p-GSK3β, T-GSK3β, and β-catenin were analyzed by Western blotting. (D) Quantification of the relative levels of p-Akt, p-GSK3β, and β-catenin (n = 3 per group, one-way ANOVA). The protein level of Fc-treated microglia served as a control. (E) Microglia from WT mice were pretreated with 25 µM of a PI3K/Akt inhibitor (LY-294002) for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 24 h. TUNEL assay was then performed and 12 different fields from three independent experiments were selected for quantifying the number of TUNEL-positive cells (one-way ANOVA). (F) Microglia from WT mice were pretreated with 10 µM SP600125, Bay 11–7082, SB203580, or U0126 for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 4 h. Real-time PCR analysis was then performed to detect the expression of IL-1β, IL-6, and TNF (n = 5 per group, one-way ANOVA). The mRNA level of DMSO- and Fc-treated microglia served as a control. (G) WT microglia were pretreated with 10 µM of a NF-κB inhibitor (Bay 11–7082) for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 1 h. The protein levels of phospho-NF-κB p65 (p-p65, Ser536) and total-NF-κB p65 (T-p65) were analyzed by Western blotting. (H) Quantification of the relative levels of phospho-NF-κB p65 (p-p65, Ser536; n = 6 per group, one-way ANOVA). The protein level of Fc-treated microglia served as a control. Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Figure 3.

sTREM2 activates the Akt–GSK3β–β-catenin and the NF-κB signaling pathways in microglia. (A) Microglia obtained from WT or Trem2-KO mice were treated with 20 nM Fc or sTREM2-Fc protein for 24 h after GM-CSF withdrawal. The protein levels of phospho-Akt (p-Akt, Ser473), total-Akt (T-Akt), phospho-GSK3β (p-GSK3β, Ser9), total-GSK3β (T-GSK3β), and β-catenin were analyzed by Western blotting. (B) Quantification of the relative levels of p-Akt, p-GSK3β and β-catenin (n = 4 per group, unpaired Student’s t test). The protein level of Fc-treated WT microglia served as a control. (C) Microglia from WT mice were pretreated with 25 µM of a PI3K/Akt inhibitor (LY-294002) for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 24 h. The protein levels of p-Akt, T-Akt, p-GSK3β, T-GSK3β, and β-catenin were analyzed by Western blotting. (D) Quantification of the relative levels of p-Akt, p-GSK3β, and β-catenin (n = 3 per group, one-way ANOVA). The protein level of Fc-treated microglia served as a control. (E) Microglia from WT mice were pretreated with 25 µM of a PI3K/Akt inhibitor (LY-294002) for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 24 h. TUNEL assay was then performed and 12 different fields from three independent experiments were selected for quantifying the number of TUNEL-positive cells (one-way ANOVA). (F) Microglia from WT mice were pretreated with 10 µM SP600125, Bay 11–7082, SB203580, or U0126 for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 4 h. Real-time PCR analysis was then performed to detect the expression of IL-1β, IL-6, and TNF (n = 5 per group, one-way ANOVA). The mRNA level of DMSO- and Fc-treated microglia served as a control. (G) WT microglia were pretreated with 10 µM of a NF-κB inhibitor (Bay 11–7082) for 30 min, followed by treatment with 20 nM Fc or sTREM2-Fc protein for 1 h. The protein levels of phospho-NF-κB p65 (p-p65, Ser536) and total-NF-κB p65 (T-p65) were analyzed by Western blotting. (H) Quantification of the relative levels of phospho-NF-κB p65 (p-p65, Ser536; n = 6 per group, one-way ANOVA). The protein level of Fc-treated microglia served as a control. Data represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

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