Chondrogenesis Regulation by ATF6 Overexpression

The micromass culture was performed as described previously [13 (link), 17 (link)]. Briefly, multipotent murine CH10T1/2 cells were trypsinized and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine serum (FBS) at a concentration of 10⁶ cells/ml, and six drops of 100 μl of cells were placed in a 60-mm tissue culture dish (BD Biosciences), respectively. After 2-h incubation at 37 °C, 1 ml of DMEM containing 10 % FBS and BMP2 protein (300 ng/ml) was added. The medium was replaced approximately every 2–3 days. To test the effect of overexpression of ATF6 protein on chondrogenesis, C3H10T1/2 cells were infected with BMP2, BMP2 + Ad-ATF6 expression adenovirus, and control green fluorescent protein (GFP) adenovirus before micromass culture.
Mouse chondrogenic ATDC5 cells were maintained in a medium consisting of a 1:1 mixture of DMEM and Ham’s F-12 medium (Flow Laboratories, Irvine, UK) containing 5 % FBS (Invitrogen), 10 mg/ml human transferrin (Roche Applied Science), and 30 nM sodium selenite (Sigma) at 37 °C in a humidified atmosphere of 5 % CO₂ in air. The ATDC5 cells were seeded at a density of 3 × 10⁵ cells/well in 6-well cell culture plates (Corning). The medium was replaced every other day. For adenovirus (Ad-ATF6 or Ad-GFP) infection and Ad-ATF6 siRNA, Ad-RFP infection, the same protocol as used with C3H10T1/2 cells was followed.