Figure 4: Lack of SGK1 activity in decidualizing cells enhances susceptibility to oxidative cell death.

(a) SGK1 mRNA expression in undifferentiated HESCs (0 d) or cultures decidualized with cAMP and medroxyprogesterone acetate (MPA) for 2–8 d. Primary cultures were established from women with RPL (n = 9) and from control subjects (n = 11). Horizontal bars indicate the median expression in each group. (b) Primary HESC cultures were first transfected with either nontargeting (NT)- or SGK1-siRNA and then decidualized for 4 d and collected for qRT-PCR or western blot analysis. Total cell lysates were probed for SGK1 and cleaved poly(ADP-ribose) polymerase-1 (PARP-1) expression. β-actin served as a loading control. (c) The oxidation status of decidualizing cells transfected with NT- or SGK1-siRNA, as determined before and after treatment with 250 μM H₂O₂ using 2′7′-dichlorofluorescein. (d) qRT-PCR analysis of cultures, treated as described in b, demonstrating that SGK1 silencing impairs the expression of GPX3 (top). Expression of GPX3 transcripts was also determined in undifferentiated and decidualizing primary HESC cultures established from subjects with RPL and control subjects (middle), as well as 8.5 d.p.c. in uteri of pregnant Sgk1^−/− female mice crossed with WT males (n = 6) and WT females with Sgk1^−/− males (n = 6; bottom). (e) Western blot analysis of total cell lysates of differentiating primary HESC cultures first transfected as indicated and then decidualized with cAMP and MPA for 4 d. (f) Primary HESC cultures, transfected as indicated, were plated in 96-well plates and decidualized with cAMP and MPA for the indicated time points. Cell viability, assessed by MTS assay, was normalized to the viability of undifferentiated cells. *P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as means ± s.e.m.