Fig. 4

Changes in the placental expression of the suppressyn-binding protein ASCT2 across pregnancy and a mechanism underlying changes in suppressyn secretion. (a)Translation levels for ASCT2, a binding protein for suppressyn, were compared between controls and preeclampsia (PE) patients throughout pregnancy. Subgroups were created based on the presence or absence of fetal growth restriction (FGR) (EO-PE-FGR(-), EO-PE-FGR(+), LO-PE-FGR(-), LO-PE-FGR(+)). Box plots display the median (horizontal center line), interquartile range (box), mean values (×) and minimum/maximum values (whiskers). The numbers in parentheses indicate the sample sizes used for each group. Statistical significance was evaluated using Mann-Whitney U tests. *p < 0.05; **p < 0.01; n.s., not significant. Quantification was performed based on results from the Supplementary Figure S2 blot, with original blot data shown in Supplementary Figure S3. (b, c) The effects of in vitro ASCT2 knockdown on suppressyn (SUPYN) secretion were examined using trophoblast cell lines: (b) JAR (trophoblast cancer cell line) and (c) HTR8 (SV/neo) - SUPYN (trophoblast-derived cell line stably expressing suppressyn). The upper panels show Western blot analysis of ASCT2 protein levels following siRNA treatment, demonstrating dose-dependent reductions. Middle panels show changes in intracellular suppressyn protein levels. The original blot data are shown in Supplementary Figure S4. The lower panels show the secreted suppressyn protein in the culture medium, as detected by ELISA. Statistical tests were performed in comparison to the 0 pmol treatment group.　All experiments were repeated three times in duplicate. Statistical significance was assessed using Mann-Whitney U testing. *p < 0.05; **p < 0.01.