Fig. 2

Estrogen promotes M2 microglial polarization through activation of STAT3. a, b Expressions of M1/M2 microglia markers were examined after the vehicle or E2 (1 nM) or E2 plus tamoxifen (1 μM) 1 treatment of human microglia (HMC3) (a) and mouse microglia (SIM-A9) (b). The value of qRT-PCR in each Figure was normalized using actin as a control. The Y-axis indicates arbitrary unit. c, d Population of M1 and M2 cells was examined by FACS after the treatment of human microglia (HMC3) with vehicle alone, E2 (1 nM) or E2 plus tamoxifen (1 μM) (c) and SIM-A9 (d) cells. e HMC3 cells were treated with or without E2 (1 nM) or tamoxifen (1 μM) for 24 h and the expressions of JAK and STAT3 were examined by western blot. f The HMC3 cells were infected with lentivirus containing green fluorescent protein (GFP) gene and GFP⁺ cells were sorted by flow cytometry (FACS). The GFP⁺ HMC3 cells were seeded in 96-well plates for 1 day and they were transfected with the Arginase-1 promoter reporter plasmid using Lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA). After 24 h, the cells were treated with only estrogen (1 nM) or estrogen plus STATTIC (0.5 μM) or tamoxifen (1 μM) and cultured for another 24 h. The expression of luciferase was measured by using IVIS Xenogen bioimager. The Arginase-1 reporter luciferase activity (M2) was normalized with the expression of GFP⁺. g, h Human microglia (HMC3) were treated with or without E2 (1 nM) and the STAT3 inhibitor (0.5 μM) or tamoxifen (1 μM) for 24 h. Cells were then subjected to flow cytometry for quantifying CD206⁺/Iba1⁺ (M2 cell) (g) and CD86⁺/Iba1⁺ (M1 cell) (h). The data are presented as the mean ± SD. One-way ANOVA, *: p < 0.05; **: p < 0.01; ***: p < 0.001. (n = 3)