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Figure 1

From: Peptide hormone ELABELA enhances extravillous trophoblast differentiation, but placenta is not the major source of circulating ELABELA in pregnancy

Figure 1

ELA and Apelin increase invasion capacity of HTR8/SVneo cells. (a) Immunohistochemistry shows ELA, APLNR and Apelin to be expressed in first trimester human placenta villous cytotrophoblasts (arrowheads) and distal column extravillous trophoblasts (open arrows). Additionally, ELA and Apelin are expressed in syncytiotrophoblasts (black arrows). No staining was observed in the rabbit IgG control staining. Antibody-specific DAB staining is shown in brown, haematoxilin counterstain was used to stain nuclei blue. (b) In HTR8/SVneo cells ELA, APLNR and Apelin are weakly expressed in adherent proliferating cells, while ELA expression becomes strong in invaded cells. No staining was observed in the rabbit IgG control staining. (c) Invasion assays show that increasing levels of ELA increase the number of invaded cells. (d) Blocking APLNR using non-peptide antagonist ML221 does not significantly reduce the effect of ELA on invasion, while ML221 itself appears to increase invasion although not significantly. (e) siRNAs targeting APLNR were tested in HTR8/SVneo cells that were stably transfected with a vector expressing APLNR. siRNA APLNR-3 and APLNR-4 were directed against untranslated regions not present in the vector and did not induce knockdown of APLNR mRNA. APLNR-1 and APLNR-2 did show knockdown of APLNR of around 80%, and were used in further experiments. (f) APLNR protein knockdown was confirmed by making use of the NanoLuc luciferase fused to APLNR in the stable transfected vector performing luciferase assays at 48, 72 and 96 hours after transfection, showing clear knockdown at 96 hours. (g) Both ELA and Apelin induce invasion of HTR8/SVneo cells, but siRNA-mediated knockdown of APLNR does not significantly reduce the effect of ELA or Apelin on invasion. Transwell invasion assay experiments were repeated 3–5 times using 3–5 replicates per treatment. Data are presented as mean ± SEM and tested with one-way ANOVA followed by Bonferroni multiple comparisons test or with Student’s t-test in case of two data sets. *Indicates p < 0.05; **Indicates p < 0.01.

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