Fig 2: Schematic model illustrating that S100P participates in trophoblast syncytialization during early placenta development by downregulating YAP1 and TEAD4.

Fig 3: Increased S100P expression during FSK-induced syncytialization of TSCT cells. (A) Quantification of S100P mRNA expression in ST(2D)-TSCT cells measured on D3 and D6. The mean values ± SEMs (normalized to GAPDH) are shown. D3: day 3, D6: day 6. *p=0.0110; ***p=0.0002; ****p<0.0001. (B) Western blotting analysis of S100P and 11β-HSD2 in ST(2D)-TSCT cells measured on D3 and D6. Semiquantitative analysis of S100P and 11β-HSD2 protein expression normalized to GAPDH (n=3). **p=0.0073; ***p=0.0006; ****p<0.0001. (C) Immunofluorescence staining of S100P (red), E-cadherin (green), β-hCG (green) and Hoechst staining (blue) in human TSCT, ST(2D)-TSCT (D6) and ST(3D)-TSCT cells (D6). White arrows indicate cytoplasmic S100P expression, and yellow arrows represent nuclear S100P expression. Representative images from three independent experiments are shown. Scale bar, 20 μm. (D) Measurement of the secreted S100P levels in human TSCT and ST(2D)-TSCT cultures (D3 and D6) by ELISA. The levels were measured in three independent experiments. ****p<0.0001. (E) Measurement of secreted β-hCG levels in human TSCT and ST(2D)-TSCT cultures (D3 and D6) as assessed by ELISA. The levels were measured in three independent experiments. *p=0.0283; ****p<0.0001.

Fig 4: YAP1 and TEAD4 expression are increased in S100P-knockdown cells and decreased in S100P-overexpressing cells. (A) Quantification of YAP1 and TEAD4 mRNA expression. ST(2D)-TSCT cells transfected with siRNA underwent syncytialization for 6 days (n=3). All the bar graphs depict the mean values ± SEMs (normalized to GAPDH), and the values were measured in duplicate; ns: p=0.0739, p=0.1537. (B) Representative Western blotting of YAP1 and TEAD4 protein levels in TSCT and ST(2D)-TSCT cells that were transfected with siS100P and underwent syncytialization for 6 days (n=3). Semi quantification of YAP1 and TEAD4 protein expression normalized to GAPDH (n=3). **p=0.0023; ***p=0.0007; ****p<0.0001. (C) Representative immunofluorescence images of staining for YAP1 and the TSCT cell boundary marker ZO-1. Hoechst staining indicates nuclei (n=3). Scale bars, 50 µm. (D) Quantification of YAP1 and TEAD4 mRNA expression was performed by qPCR. TSCT cells were infected with a lentiviral vector inducing overexpression of S100P (n=3). All the bar graphs show the mean values ± SEMs (normalized to GAPDH). ns: p=0.1643, p=0.0796. (E) Representative Western blotting of YAP1, TEAD4, and S100P expression in S100P-overexpressing TSCT cells. Semi quantification of YAP1, TEAD4 and S100P protein expression normalized to GAPDH (n=3). **p=0.0018; ***p=0.0006; ****p<0.0001. (F) Representative immunofluorescence images of ZSGreen-S100P and YAP1 staining. Hoechst staining indicates nuclei. Scale bars, 50 µm. (G) Representative Western blotting of YAP1 and TEAD4 expression in TSCT cells treated with recombinant human S100P protein (5 µM, 10 µM, 15 µM, and 20 µM) (n=3). Semi quantification of YAP1 and TEAD4 protein expression normalized to GAPDH (n=3). *p=0.0151, p=0.0126, p=0.0203; **p=0.0051; ***p=0.0005, p=0.0001; ****p<0.0001.

Fig 5: Placenta and serum S100P levels in healthy controls and RPL patients. (A) Immunofluorescence staining of S100P (green), 11ß-HSD2 (red), ß-hCG (red) and Hoechst staining (blue) in human healthy placentas (sixth to eighth week of gestation). White arrows indicate STB. Representative images from three independent experiments are shown. Scale bar, 50 µm. (B) Immunohistochemistry staining of S100P in human placentas extracted from women at six- to-eight weeks of gestation. ß-hCG was used to identify syncytiotrophoblast layers. Scale bars, 50 µm. (C) Representative Western blotting of S100P expression in normal and RPL placental tissues (Ctrl n=6 vs. RPL n=7). (D) Measurement of serum S100P levels in healthy controls and RPL patients by ELISA (n=16). **p=0.0079.