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Fig. 2 | Reproductive Biology and Endocrinology

Fig. 2

From: Estrogen-sensitive activation of SGK1 induces M2 macrophages with anti-inflammatory properties and a Th2 response at the maternal–fetal interface

Fig. 2

E2 upregulates SGK1 activity in THP-1 monocyte-derived macrophages via ERβ. A Representative immunofluorescence staining of THP-1 monocyte-derived macrophages before (resting phenotype, top), and after (differentiated phenotype, bottom) PMA incubation. The differentiation of macrophages was confirmed by staining with the antibodies against macrophage-specific CD68 (green) and with DAPI (blue) for nuclei. Final magnification: × 630, scale bar 25 μm. B Western blotting analysis of THP-1 monocyte-derived macrophages treated with E2 (10 nM) alone, E2 plus ER antagonist ICI182780 (1 μM), E2 plus ERα antagonist MPP (1 μM) and E2 plus ERβ antagonist PHTPP (1 μM) for 24 h. Blots were probed with antibodies to t-SGK1, p-SGK1, and β-actin. C Densitometric quantifications of the arithmetic mean (SEM) ratio of p-SGK1 (left), t-SGK1 (middle), and p/t-SGK1 protein (right) to β-actin in THP-1 monocyte-derived macrophages. Western blotting analysis (D) and densitometric quantifications (E) of THP-1 monocyte-derived macrophage lysates pretreated with an ESR2 (encoding ERβ)-specific siRNA and non-targeting (NT) siRNA. Blots were probed with antibodies to p-SGK1 (left), t-SGK1 (middle), and p/t SGK1 protein (right) in THP-1 monocyte-derived macrophage lysates. β-actin was the loading control. F Blots were probed with antibodies to ESR2 and β-actin prepared from lysates of cell pretreated with NT siRNA or ESR2 siRNA. Three independent samples were analyzed. Data are expressed as the arithmetic means ± SEM. **P < 0.01, ***P < 0.001, contrasted with control or medium group; ∆∆∆P < 0.001, contrasted with E2 group; ###P < 0.001, contrasted with the E2 + ICI 182780 or E2 + NT siRNA group; +  +  + P < 0.001, contrasted with the E2 + PHTPP group. E2, estradiol; SGK1, serum-glucocorticoid regulated kinase; ER, estrogen receptor; PMA, phorbol 12-myristate 13-acetate; CD68, CD68 molecule; DAPI, 4′,6-diamidino-2-phenylindole; NT, non-targeting; siRNA, small interfering RNA; SEM, Standard Error of the Mean

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