Fig 1: Downregulation of PCBP2 severely affects first trimester placental explant outgrowth. (A) Representative images of first trimester placental explants treated with siRNAs targeting PCPB1, PCBP2, YBX1, or non-targeting siRNAs that developed EVT outgrowth. (B) Number and percentage of explants that showed EVT outgrowth out of the total number of explants that were treated with the different siRNAs. PCBP2 downregulation leads to the strongest inhibition of explant outgrowth amongst the genes tested. (C) Representative images of immunohistochemical staining for PCBP1, PCBP2, and YBX1 proteins in first trimester placental explants transfected with the different siRNAs. The stainings demonstrate successful downregulation of the respective proteins. No staining was observed in mouse IgG control staining. Antibody-specific DAB staining is shown in brown, hematoxylin counterstain was used to stain nuclei blue. (D) Representative phosphoH3(Ser10) and HLA-G immunohistochemical stainings of first trimester placental explants after transfection with PCBP1, PCBP2, YBX1, or non-targeting siRNAs to identify proliferating cells undergoing mitosis and to indicate the location of EVTs, respectively. (E) Quantification of the ratio of H3/HLA-G representing proliferating EVTs indicates a significant induction of proliferating EVTs upon PCBP2 knockdown while YBX1 silencing leads to a reduction of proliferating EVTs. Within the different conditions each dot represents an explant with quantifiable outgrowths. In total this analysis was performed on explants derived from five different first trimester placentas. Data are presented as mean ± SD and tested with one-way ANOVA followed by Dunnett’s multiple comparisons test. *Indicates p < 0.05.