Fig 1: Protein levels of CCN3 and mCCN3 isoforms in human osteosarcoma cell lines. Western blotting examination of CCN3 and mCCN3 isoforms and corresponding band intensity in nuclear fraction (A–C) and cytoplasmic fraction (D,E), compared with MG63 (t-test), and in secreted fraction (F,G) compared with mM or compared with mS (t-test). mM: culture medium for MG-63 cells; mS: culture medium for Saos-2 cells. **p < 0.01.
Fig 2: Immunolocalization of CCN3, p16, p21, and Cyclin D1 in EVT cells of late AIP. Double immunolabelling of CCN3 (A–C), p16 (E–G), p21 (I–K), and Cyclin D1 (M–O), in green, with HLA-G, in red; blue, DAPI, respectively. Red arrow, nuclear expression; green arrow, membrane/cytoplasmic. Negative controls were performed by omitting the primary antibody (D, H, L, P). Scale bar represents: (A–P), 100 µm. The red box is an enlarged part of the white dotted box.
Fig 3: Schematic overview of proposed CCN3-mediated signaling pathways in placental diseases PE and AIP. This figure summarizes the results of this study and showed the proposed signaling pathway of CCN3 in placentas of PE and AIP diseases. In early-onset preeclamptic placentas, CCN3 may promote STB and CCT cell cycle progression via p21/Cyclin E1/pRB and FAK/Akt/mTOR pathway. We speculate that the invasion and migration capacities of the EVT may be decreased by subsequently inhibiting FAK/Akt pathway and cleaved Notch/p21 signaling. Increased p53 expression can induce cell apoptosis and decreased p21 that also inducing cell apoptosis, which may result in PE. However, in the late AIP group, CCN3 may mediate cell cycle arrest by bringing about senescence which is advantageous for the differentiation from CTB to STB and EVT. Furthermore, CCN3 may enhance invasion and migration capacities of the trophoblast by activating FAK/Akt pathway. Although an increase in p53 may increase the apoptosis of trophoblast cells, increased p21 may also promote cell viability through an anti-apoptotic pathway, which may contribute to the AIP disease. CCT, cell column trophoblast; EVT, extravillous trophoblast; STB, syncytiotrophoblast.
Fig 4: Immunolocalization of CCN3, p16, p21, and Cyclin D1 in villous trophoblast cells of preeclamptic placenta. Double immunolabelling of CCN3 (A, B), p16 (D, E), p21 (G, H), and Cyclin D1 (J, K), in green, with the trophoblast marker CK-7, in red; blue, DAPI, respectively. Red arrow, nucleus expression; green arrow, membrane/cytoplasmic expression. Analysis of representative early control and early preeclamptic placental sections. Negative controls were performed by omitting the primary antibody (C, H, L, P). Scale bar represents: (A–L), 100 µm. The red box is an enlarged part of the white dotted box.
Fig 5: Effect of overexpressed CCN3 and mCCN3 on MG-63 cell invasion. (A–E) MG-63 cells were infected with lentiviruses overexpressing CCN3 and mCCN3. The cells were subjected to qRT-PCR analysis of CCN3 (A), COX-2 (C), MMP2 (D), and MMP13 (E) and to trans-well assays (B). Con: pLenti6.3-V5 vector control; CCN3: pLenti6.3-CCN3 plasmid expressing human CCN3 coding sequence; mCCN3: pLenti6.3-mCCN3 plasmid expressing human mCCN3 coding sequence. *p < 0.05, **p < 0.01 compared with Con (t-test).
Supplier Page from Abcam for Anti-CCN3 antibody