Fig 2: Cdc5l promotes early chondrocyte differentiation.A, ATDC5 cells were transfected with siCont or siCdc5l overnight, induced with BMP-2 + ITS for the indicated time points, and subjected to immunoblotting for Cdc5l. Tubulin served as a loading control. B, ATDC5 cells were stimulated with or without BMP-2 + ITS for 4 days and subjected to RT-qPCR for miR-542-3p (n = 3). C, ATDC5 cells, primary (pr.) chondrocytes, or C3H/10T1/2 cells transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS and subjected to alcian blue staining on day 8, 13, or 16, respectively. D and E, ATDC5 cells (D) or primary (pr.) chondrocytes (E) transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS and subjected to RT-qPCR analysis for Sox9 and Col2a1 on day 7 or 6, respectively (n = 3). F and G, C3H/10T1/2 cells transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS for 10 days and subjected to RT-qPCR for Sox9 and Agc1 (F) or Scx and Tnc (G) (n = 3). H, primary (pr.) mesenchymal stem cells (MSCs) transfected with siCont or siCdc5l were stimulated with or without TGF-ß3 and a cocktail of dexamethasone, ascorbic acid, and ITS (DAI), and subjected to RT-qPCR analysis for Sox9 and Col2a1 on day 6 (n = 3). I, primary (pr.) chondrocytes transfected with siCont or siCdc5l were stimulated with or without BMP-2 + ITS and subjected to RT-qPCR analysis for Col10a1 on day 6 (n = 3). J, ATDC5 cells transfected with siCont or siCdc5l were infected with AAV-LacZ or AAV-CDC5L for 2 days, followed by BMP-2 + ITS application for 4 days. RT-qPCR for Sox9 and Col2a1 was analyzed (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.

Fig 3: An illustration of this study's results and a working model. CDC5L is expressed in PCNA-positive proliferating and differentiating chondrocytes but not in hypertrophic chondrocytes. CDC5L-mediated cell cycle (G2/M) progression is essential for conversion of resting chondrocytes into proliferating chondrocytes to accelerate cell growth to enlarge the cartilage template of ossification. The inhibition of pre-mRNA splicing and subsequent expression of WEE1 by CDC5L plays a substantial part in this cell cycle acceleration. Hence, expression of CDC5L and WEE1 is mutually exclusive. Meanwhile, CDC5L promotes early chondrocyte differentiation by enhancing pre-mRNA splicing and subsequent expression of SOX9 and COL2A1. CDC5L has dual roles in early chondrogenesis, promoting cartilage growth by WEE1 suppression and enhancing early differentiation through upregulation of SOX9 and COL2A1 via finely tuning the splicing of the target pre-mRNAs.