Atdc5

The mouse chondrogenic cell line, ATDC5, was obtained from the RIKEN Cell Bank. The human juvenile costal chondrocyte cell line, C28/I2, was provided by Dr. W U Kim (Catholic University, Korea). HEK293-TOP cells (HEK293 cells containing the chromosomally incorporated TOPFlash gene) were provided by Dr. S Oh (Kuk Min University, Korea). ATDC5 cells were maintained in DMEM/F12 (1:1) (Gibco) supplemented with 5% FBS (Gibco). To induce hypertrophic differentiation, ATDC5 cells were incubated with insulin–transferrin–sodium selenite supplement (Gibco) in three-dimensional alginate beads for 3 d, as described previously (Kawasaki et al, 2008 (link)). C28/I2 and HEK293-TOP cells were maintained in DMEM (Gibco) containing 10% FBS. All chemicals were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) for the in vitro studies. For E₂ (17β-estradiol; Sigma-Aldrich) treatment, the cells were cultured in phenol red–free DMEM/F12 with 5% charcoal-stripped FBS for 24 h followed by serum-free medium for 24 h before the experiment. The PTD-DBMP was synthesized by Peptide 2.0 Inc.

Murine chondrocyte cell line ATDC5 cells were purchased from the Riken Cell Bank (Tsukuba, Japan). The cells were maintained in Dulbecco’s minimal essential medium nutrient mixture F-12 HAM (DMEM/F12; Sigma) containing 5% fetal bovine serum (FBS; Sigma) in 5% CO₂ in air. For chondrocyte differentiation, ATDC5 cells were treated with chondrogenic medium (5% FBS in the presence of ITS) with or without 10μM RO-3306 (Sigma) to inhibit Cdk1 activity, or 3 × 10⁻⁷ M recombinant PTHrP (Peptide Institute, Inc) to activate PTHrP signaling. Results are representative of more than four individual experiments.

A murine chondrogenic cell line, ATDC5, was purchased from the RIKEN Cell Bank (Tsukuba Science City, Japan). ATDC5 cells were cultured at a density of 1 × 10⁴ cells/cm² in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (Gibco/BRL, Gaithersburg, MD, USA) containing 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), followed by replacement with DMEM/F12 containing 5% FBS, 10 μg/mL human recombinant insulin (Wako Pure Chemical, Osaka, Japan), 10 μg/mL transferrin (Roche Diagnostics, Mannheim, Germany) and 3 × 10⁻⁸ M sodium selenite (Sigma) for the promotion of cell differentiation. The cells were then cultured at 37 °C for different periods up to 12 days under 5% CO₂. RNA was extracted from the cultured ATDC5 cells when they became confluent (4 days after plating) and was then extracted every 2 days after confluence.
Day-10 cultures of ATDC5 cells were treated with IL-1β (10 ng/mL, R&D Systems, Minneapolis, MN, USA), 100 nM all-trans-retinoic acid (ATRA; Sigma, St. Louis, MO, USA), the 100 nM RARγ selective agonist AGN204647 [7 (link)], the 100 nM RARα-selective agonist AGN195183 [48 (link)], the 100 nM RAR inverse agonist AGN194310 [7 (link)], the selective inhibitor of ERK1/2 kinase PD98059 (20 μM, Calbiochem, La Jolla, CA, USA), the selective inhibitor of p38 kinase SB203580 (20 μM, Calbiochem), or combinations of these agents for 24 h.