Atdc5

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.

Raw264.7 macrophages from the American Type Culture Collection (ATCC, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4.5 g/L; Gibco, USA), containing 100 U/mL penicillin, 100 mg/mL streptomycin sulfate (Life Technologies, USA), and 10% FBS (Gibco, USA). The pre-chondrocyte cell line ATDC5 (Tsukuba, Japan) was maintained in DMEM/F12 (Gibco, USA), containing 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, 5% FBS, and 1× ITS universal culture supplement premix reagent (BD Biosciences).

The chondrogenic cell line (ATDC5, Sigma Aldrich, Cat # 99072806) was used for all in vitro studies. Cells were used at passage 5 and under for all experiments. ATDC5 cells were maintained using media DMEM/F12 (Thermo Fisher, Cat# 11320033), 5% fetal bovine serum (FBS, Thermo Fisher, Cat# 16000044) and 1% penicillin/streptomycin (P/S, Thermo Fisher, Cat# 15140122). For all in vitro experiments, ATDC5 cells were plated at 20,000 cells/well in 12 well plates. Prior to transfection experiments, chondrocytes underwent serum starvation using basal media OPTI-MEM (Thermo Fisher, Cat# 31985070) 0.5% FBS and 1% P/S for 24 h. All transfection protocols were executed using serum-free media, OPTI-MEM supplemented with 1% P/S. The cytotoxic effects were assessed using PrestoBlue™ Cell Viability Reagent (Thermo Fisher, Cat# A13261) according to the manufacturer’s protocol. To quantify the number of metabolically active cells, a standard curve of ATDC5 cells was created and the absorbance values were related back to the number of cells for quantitation. The plate was read at 570 nm with a reference wavelength at 600 nm using a plate reader (TECAN infinite M200 Pro).

As mentioned earlier [24 (link)], mouse primary chondrocyte cell line (ATDC5) and macrophage cell line (RAW264.7) were bought from Sigma Aldrich (St. Louis, MO). ATDC5 and RAW264.7 cells were grown in the DMEM/F12 medium (Thermo Fisher Scientific, Shanghai, China) comprising 5% fetal bovine serum (Thermo Fisher Scientific, Shanghai, China). The cells were maintained at 37°C in a humidified incubator with 5% CO₂, with the medium altered every two days. ATDC5 cells were treated with varying concentrations of IL-1β (5, 10, 20, 100 ng/mL) for 24 hours. RAW264.7 cells were treated with LPS (100 ng/mL) + IFNγ (20 ng/mL) and IL-4 (20 ng/mL) + IL-13 (20 ng/mL) for 24 hours, respectively.
After RAW264.7 macrophages were treated with LPS (100 ng/mL) +IFN-γ (20 ng/mL) for 24 hours to induce M1-polarized macrophages, the supernatant of polarized macrophages was harvested via centrifugation (1000 rpm, 5 minutes) and preserved at −80°C for further experiments. Conditioned medium (CM) from differentiated macrophages was diluted with a serum-free medium at a ratio of 1:1 and added to chondrocytes for further culture. LPS (Escherichia coli 0111:B4) was bought from Sigma-Aldrich (St. Louis, MO), while IL-1β, IL-4, IL-13 and IFN-γ were acquired from PeproTech (Rocky Hill, NJ, USA).