Fig 2: Overexpression of CYP11A1 induces androgen production and syncytiotrophoblast formation in BeWo cell line(A) Overexpression of CYP11A1 and forskolin (10 µM) treatment increase pregnenolone, progesterone and testosterone secretion in BeWo cell line. (B, C) E-cadherin staining show that overexpression of CYP11A1 could reduce E-cadherin staining intensity, indicating syncytiotrophoblast formation in BeWo cell line(60×). Cox-IV was used as mitochondrial marker. * means P < 0.05, ** means P < 0.01, *** means P < 0.001. FSK = foskolin, 3.1 = control plasmid (pcDNA-3.1), CYP = CYP11A1 over expression plasmids (pcDNA-3.1-CYP11A1).

Fig 3: CYP11A1 overexpression compromise trophoblast invasion and inhibit MMP-2 expression(A) The picture shows that trophoblast invasion could be inhibited by CYP11A1 overexpression, which could be rescued by flutamide treatment. (B) Testosterone treatment could inhibit trophoblast invasion. (C) Western blot result of MMP-2 shows that overexpression of CYP11A1 could inhibit MMP-2 expression, which could rescued by androgen receptor (AR) inhibitor, flutamide. The statistic result is shown in the right panel. * means P < 0.05, ** means P < 0.01, *** means P < 0.001,****means P < 0.0001.

Fig 4: SVIP modulates the expression levels of several proteins related to steroidogenesis. (A) After H295R cells were transfected with 0.75 µg SVIP, mRNA levels of CYP17A1, CYP11A1, CYP11B1, HSD3ß2, LDLR, HMGR, CYP21A2, and SVIP were examined by RT-qPCR. The experiment was repeated at least three times with three replicates. Error bars represent standard error (*p < 0.05 and ***p < 0.001) (B and C) Protein expression levels of CYP17A1, CYP11A1, CYP11B1, HSD3ß2, LDLR, StAR, HMGR, and SVIP were determined by IB of cells transfected with (B) 0.5 and 1.5 µg plasmid encoding SVIP or (C) SVIP siRNA. (D) Cells were transfected with 1.5 µg SVIP and then treated with cycloheximide for the indicated times. CYP17A1 and SVIP levels were determined via IB. The degradation rates of substrates were calculated using three independent experiments (*p < 0.05). GAPDH was used as loading control.

Fig 5: Levels of steroidogenic enzymes in in vivo PE models. (A) The expression of steroidogenic enzymes in the L-NAME, COMT-I, and RUPP groups was analyzed by western blot analysis. (B and C) The levels of CYP11A1, CYP17A1, HSD3B1, HSD17B3 and CYP19A1. The individual protein expression was normalized to that of ß-actin. Data are expressed as the mean ± SD. *P<0.05 vs. CON group; #P<0.05 vs. Sham group. L-NAME, N-nitro-L-arginine methyl ester hydrocholride; COMT-I, catechol-o-methyltransferase inhibitor; RUPP, reduced uterine perfusion pressure; CYP11A1, cholesterol side-chain cleavage enzyme; CYP17A1, 17a-hydroxylase/17,20-lyase; HSD3B1, 3ß-hydroxysteroid dehydrogenase/d5 4-isomerase type 1; HSD17B3, 17ß-dehydrogenase 3; CYP19A1, aromatase cytochrome P450; CON, control.