Cartilage homeoprotein 1 (CART-1) is a homeoprotein which has been suggested to play a role in chondrocyte differentiation and in skeletal development. It is expressed mainly in prechondrocytic mesenchymal condensations. Patients with mutations in the CART-1 gene display several craniofacial abnormalities, suggesting that CART-1 has a functional role in craniofacial skeletal development. However, its target genes and position in the established chondrogenic pathways is poorly documented. Given the fact that CART-1 is expressed predominantly in the chondrocyte lineage and its role in skeletal development, we hypothesized that CART-1 regulates expression of several pivotal genes involved in chondrogenic differentiation. The coding sequence of human CART-1 was custom synthesized with optimized codon usage and cloned into a p3XFLAG-CMV-7.1 expression vector. FLAG-CART-1 was transiently overexpressed in SW1353 cells by polyethyleneimine-mediated transfection (1,000 ng of plasmid/well in 12-well plates). FLAG-Empty vector was used as a negative control. FLAG-CART-1 overexpression was confirmed by means of anti-FLAG immunoblotting. To investigate a potential connection between CART-1 and established key chondrogenic pathways, TGFβ3 (10 ng/mL) was added to SW1353 cells in CART-1 overexpression cultures or their appropriate controls. Cells were harvested 48 hours after transfection and mRNA expression of several genes involved in chondrogenic differentiation was determined by qRT-PCR. Data represent three separate experiments performed in technical triplicate.Introduction
Methods
HACs from five OA patients (passage 1) were cultured in cytokine-free medium, under 280 or 380 mOsm respectively, under standard 2D Introduction
Methods
