Fig 2: RSK2 upregulates cyclin D1 via promoting FOXO1 degradation. (A) Western blot was used to measure FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (B) RT-qPCR was used to measure FOXO1 mRNA expression in A375 cells transfected with RSK2 siRNA. (C) The influence of RSK2 on FOXO1 degradation was estimated in A375 cells under MG132 treatment. (D) The effect of RSK2 on FOXO1 protein stability was evaluated in A375 cells under CHX treatment for the indicated time. (E) HEK293T cells were co-transfected with Flag-FOXO1 and HA-RSK2 plasmid as indicated, immunoprecipitation with anti-Flag antibody was performed. (F) Western blot was used to measure p-FOXO1 (S319) and FOXO1 expression in A375 cells transfected with RSK2 siRNA or HA-RSK2 plasmid. (G) Western blot was used to measure cyclin D1 expression in A375 cells co-transfected with Flag-FOXO1 and HA-RSK2 as indicated. (H) A schematic model of RSK2–FOXO1–cyclin D1 axis leading to melanoma proliferation and vemurafenib resistance.

Fig 3: Working model of E2-activated SGK1 involvement in negative regulation of TLR-4-mediated apoptosis and pro-inflammatory TH1 immune responses at the feto-maternal interface. E2-activated SGK1 via the PI3K signaling pathway promoted cell viability and suppressed LPS-induced apoptosis of DSCs by up-regulating the expressions of anti-apoptotic genes, and attenuating pro-apoptotic FOXO1 activation. E2-sensitive activation of SGK1 down-regulates TLR4-mediated NF-κB activation, leading to reduced production of pro-inflammatory cytokines and promoting TH2 shift at the feto-maternal interface, which is beneficial to successful gestation.

Fig 4: E2-stimulated activation of SGK1 attenuates TLR4-mediated apoptosis of DSCs. (A) Relative efficiencies of DSC growth were assessed with the MTT assay. (B) TLR-4-triggered apoptosis of E2-pretreated DSCs in the presence or absence of SGK1 inhibitor GSK650394 was determined by flow cytometry based on PI staining and annexin expression. Transcript levels the of anti-apoptotic genes BCL2 and XIAP (C), and the decidual marker gene PRL (D) in LPS-stimulated DSCs incubated with E2 with or without GSK650394. Protein levels of BCL2 (E), XIAP (F) and PRL (G) were analyzed by immunoblotting in LPS-stimulated DSCs incubated with E2 with or without GSK650394. (H) Western blot analysis of cell lysates of LPS-stimulated DSCs pretreated with E2 with or without GSK650394. Blots were probed with antibodies to total-FOXO1 (t-FOXO1), phosphorylated-FOXO1 (p-FOXO1) and β-actin as a loading control. (I) Densitometric quantification of phosphorylated- and total FOXO1 to β-actin (left), and mean (SEM) ratio of phosphorylated-to-total protein for FOXO1 (right). Control group, phenol red-free RPMI-1640 media; LPS group, 10 ng/mL LPS; LPS+E2 group, 10 ng/mL LPS + 10 nM E2; LPS+E2+GSK650394 group, 10 ng/mL LPS + 10 nM E2 + 10 μM GSK650394. Data are represented as arithmetic means ± SEM for 3 individual DSCs from early normal pregnancy. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group; ∆P < 0.05, ∆∆P < 0.01, ∆∆∆P < 0.001, compared with LPS group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with LPS+E2 group.

Fig 5: Schematic model of GLK overexpression-induced attenuation of Foxp3 transcription. GLK overexpression in Treg cells phosphorylates IKKβ at Ser733 residue in a PKCθ-independent manner. The transcription factor FoxO1 binds to the Foxp3 promoter and promotes Foxp3 transcription in normal Treg cells. The GLK-phosphorylated IKKβ interacts with and induces FoxO1 Ser319 phosphorylation, leading to FoxO1 nuclear export and Foxp3 downregulation. Thus, GLK-IKKβ-FoxO1 signaling attenuates differentiation and function of Treg cells.