Fig 2: 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.

Fig 3: Identification of PCBP2 as a cGAS-interacting factor.a HEK293T cells were co-transfected with Myc-tagged cGAS and Flag-tagged PCBP2 or an empty vector, and cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblot analysis. b, c Purified cGAS-His was incubated with GST-PCBP2 or GST-GFP and then pulled down with Ni-Sepharose beads (b) or glutathione-Sepharose beads (c). Western blotting was performed to detect the presence of His-tagged cGAS and GST-tagged PCBP2 proteins. d THP-1 cells were mock-infected or infected with HSV-1 for the indicated times, and then cell lysates were immunoprecipitated with a rabbit anti-cGAS antibody or control IgG, followed by immunoblotting. e HeLa cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, the cells were fixed, stained with DAPI, and observed by confocal microscopy. Scale bars, 10 µm. f Schematic diagram of PCBP2 full-length (FL) and its truncated mutants (upper panels). HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting (lower panels). g Schematic diagram of cGAS and its truncated mutants (upper panels). HEK293T cells were co-transfected PCBP2 with cGAS, its truncated mutants, or an empty vector, as indicated. The cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting with the indicated antibodies (lower panels). Source data are provided as a Source data file.

Fig 4: Schematic diagram of the mechanism underlying CCT3 stimulation of YAP and TFCP2 in liver cancer.Briefly, PCBP2 interacts with YAP directly via the WW-WB interaction and interacts with TFCP2 indirectly via the aid of YAP through the WW-PSY interaction. The ubiquitination of YAP and TFCP2 was mediated by their interactions with PCBP2. However, CCT3 prevents PCBP2 from binding with YAP and TFCP2, thereby reducing their ubiquitination and increasing their stability. Via such effects, the protumourigenic functions of YAP and TFCP2 in liver cancer are enhanced

Fig 5: CCT3 blocked the interaction between PCBP2 and YAP/TFCP2.a PCBP2 directly interacted with YAP but not with TFCP2, as evaluated by PLA in Bel-7402 and SMMC-7721 cells. Scale bar, 50 μm. b YAP, TFCP2 bond with PCBP2, as measured by co-IP in Bel-7402 cells. c Schematic presentation of the hypothesis that CCT3 might prevent YAP/TFCP2 from interaction with PCBP2. d Interaction between exogenous PCBP2 and YAP, but not between PCBP2 and TFCP2, was identified by co-IP in Bel-7402 cells. e The WB2 motif was essential for the interaction between PCBP2 and YAP, as measured by co-IP in Bel-7402 cells. d, deletion. f The PCBP2-YAP interaction replied on the two WW domains in YAP, as measured by co-IP in Bel-7402 cells. d, deletion. g The WB2 motif was critical for ubiquitination of YAP and TFCP2. h The WB2 motif of PCBP2 was essential for CCT3-mediated suppression of ubiquitination of YAP and TFCP2, as measured in Bel-7402 cells with or without expressing increasing concentrations of CCT3. i The two WW domains of YAP were prerequisite for suppression of TFCP2 by PCBP2, as measured in control cells, Bel-7402 cells with YAP knocked down with or without simultaneous expressing exogenous YAP-WT/YAP-d2WW. j The two WW domains of YAP were essential for CCT3-induced disassociation of PCBP2 from YAP/TFCP2, as measured by co-IP in Bel-7402 cells. To clearly identify the interaction between PCBP2, TFCP2 and YAP, we artificially adjusted the concentrations of PCBP2 to the same level in the immune precipitates that were immunoprecipitated by the anti-PCBP2 antibodies. Images of WB are representative of three independent experiments