Human transferrin

Cells (1 × 10⁵ cells/well) at 10 days after retroviral transduction were seeded to Ultra-Low Attachment Surface 6 well plates (Corning Inc., Corning, NY, USA) in a serum-free DMEM medium containing 10 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan), 10 mg/mL human insulin (CSTI, Miyagi, Japan), 100 mg/mL human transferrin (Roche, Basel, Switzerland), and 100 mg/mL bovine serum albumin (BSA; Nacalai Tesque) and incubated at 37 °C in a 5% CO₂ incubator for 10 days [13 (link)]. Tumor spheroids were manually counted under an inverted phase contrast microscope (BZ-X710 Microscope and BZ-X Viewer, BZ-X Analyzer imaging system, Keyence, Osaka, Japan). All morphometric studies were performed by two examiners blinded to treatment conditions.

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.

The C3H10T1/2 cell line was obtained from the RIKEN cell bank (Tsukuba, Japan). Cells were maintained in Dulbecco’s modified Eagle’s medium (GIBCO, Tokyo, Japan), supplemented with 10% fetal bovine serum, in polystyrene dishes at 37°C under 5% CO₂. Differentiation medium was prepared by supplementing the maintenance medium with 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO), 0.17 mM ascorbic acid (Sigma-Aldrich), 10 μg/ml human transferrin (Roche Molecular Biochemicals, Mannheim, Germany), 3.0 × 10^-8 M sodium selenite (Sigma-Aldrich). Maintenance medium or differentiation medium was changed twice a week without damaging the gels.