Fig. 5
E2-activated SGK1 negatively regulates NF-κB to compromise the secretion of pro-inflammatory cytokines by macrophages. THP-1 monocyte-derived macrophages stimulated by LPS were pretreated with E2 in the presence or absence of GSK650394 (SGK1 inhibitor) and BAY11-7082 (NF-κB inhibitor). Whole-cell lysates, cytosol and nucleus lysates, and cell-free supernatants were collected to assess the cytokines levels and NF-κB activities, respectively. A TLR4-mediated production of pro-inflammatory cytokines TNF-α, IL-6, and IL-12p70 of THP-1 monocyte-derived macrophages pretreated with E2 in the presence or absence of GSK650394 and BAY11-7082 was determined using the ProcartaPlex® Multiplex Immunoassay. B Western blot of whole cell lysates probed for p-IκBα, t-IκBα, and total β-actin. C The ratio of phospho-to-total proteins for IκBα (right) and relative protein levels of p-IκBα and t-IκBα to β-actin (left) were determined using densitometry. D Blots probed with antibodies for p-NF-κB, t-NF-κB, and total β-actin. E The ratio of phospho-to-total proteins for NFκB (right), and the relative protein levels of p-NF-κB and t-NF-κB to β-actin (left) were determined using densitometry. F Cytosol lysates probed for cytosolic NF-κB p65 and β-actin (left), and densitometric quantification of the ratio of the cytosolic level of NFκB p65 to β-actin (right). G Nuclear lysates probed for nuclear NF-κB p65 and TBP (left), and the densitometric quantification of the ratio of the nuclear level of NFκB p65 to TBP (right). Blots probed for SGK1 (H), cytosolic (I) and nuclear (J) NF-κB p65 of lysates of macrophages following non-targeting (NT) or SGK1-specific RNA silencing and then stimulated with E2. Data are the arithmetic means ± SEM for three biological replicates. **P < 0.01, ***P < 0.001, contrasted with the control or medium group; ∆∆P < 0.01, ∆∆∆P < 0.001, contrasted with the LPS group or E2 group; ###P < 0.001, contrasted with the LPS + E2 group or E2 + NT siRNA group; + + + P < 0.001, contrasted with the LPS + E2 + GSK650394 group. E2, estradiol; SGK1, serum-glucocorticoid regulated kinase; NF-κB; nuclear factor kappa B; TNF, tumor necrosis factor; IL, interleukin; p-, phospho-; t-, total-; IκBα, inhibitor of nuclear factor kappa-B kinase subunit alpha; TBP, TATA binding protein; SEM, Standard Error of the Mean; GSK, GSK650394; BAY, BAY11-7082