Streptomycin

Mouse CDCs were expanded using methods similar to those previously described [23 (link)]. Briefly, atria from mice were minced into small fragments and cultured as “explants” on dishes coated with 15 μg/ml fibronectin (CORNING), by using IMDM basic medium (Gibco), supplemented with 10% fetal bovine serum (HyClone), 100 units/ml penicillin G and 10 μg/ml streptomycin (WAKO, Japan). Stromal-like flat cells and phase-bright round cells emerged from the tissue fragments in 3–5 days and became confluent within 2 weeks. Twice-passaged CDCs were used for the experiments.
Mouse embryonic fibroblasts (MEFs) were purchased from company (EmbryoMax® PMEF-P3, strain CF-1, Millipore, Billerica, MA) and cultured on 0.1% (w/v) gelatin-coated culture dishes in high glucose DMEM medium (Wako, Japan), supplemented with 10% fetal bovine serum, 100 units/ml penicillin G and 10 μg/ml streptomycin.
Totally confluent CDCs or MEFs were changed with fresh medium, and conditioned media were obtained 24 hours later. All cells were cultured in a 5% CO₂ incubator at 37°C.

The mouse fibroblast-like cell line L929 (ECACC 85011425, KAC Co., Ltd., Kyoto, Japan) was cultured on collagen-coated PDMS with various stiffness. L929 cells were grown in the L929 growth medium consisting of Dulbecco’s modified Eagle medium/Ham’s F12 (DMEM/F12, Gibco, New York, USA) supplemented with 7% fetal bovine serum (FBS) (Japan Biocealum Co., Ltd., Hiroshima, Japan), 100 U of penicillin, and 100 μg/mL of streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37 °C in a 5% CO₂ atmosphere. The cells were then passaged twice. After 80–90% confluences, the cells were detached with 0.25% trypsin/1 mM Ethylene Diamine Tetra-acetic Acid (FUJIFILM Wako Pure Chemical Corporation) and seeded on collagen-coated or non-coated substrates of a 12- or 24-well culture-graded polystyrene plate or soft, mid, or hard PDMS at 1.6 × 10⁴ or 1.0 × 10⁵ cells/cm², respectively. The cells were cultured for up to 3 days at 37 °C in a 5% CO₂ atmosphere, and the medium was renewed every three days.
The culture supernatants of L929 cells cultured for 12 h on each substrate were collected and filtered through a Millex-Gp 0.22 µm membrane filter (Merck Millipore Ltd., Carrigtwohill, Ireland) into a tube. The supernatants were stored at –80 °C prior to use in the subsequent osteoclast precursor cell experiment.

PASMCs (passages 5–10) from normal subjects (Lonza, Basel, Switzerland) and IPAH patients (kindly offered by Prof. Jason X.-J. Yuan) (18 (link), 19 (link)) were cultivated in Medium 199 supplemented with fetal bovine serum (FBS, 10%; Thermo Fisher Scientific, Waltham, MA, USA), D-valine (50 μg/ml; MilliporeSigma, Burlington, MA, USA), endothelial cell growth supplement (20 μg/ml; BD Biosciences, Franklin Lakes, NJ, USA), penicillin G (100 U/ml), and streptomycin (100 μg/ml; Fujifilm Wako Pure Chemical, Osaka, Japan) at 37°C.

Caco-2 cells (ATCC, HTB-37) were cultured in MEM basic medium (Gibco) supplemented with Earle’s salts, L-glutamine and 20% fetal bovine serum. After detachment with 0.25% Trypsin-EDTA (Gibco), the cells were cultured in a 96-well plate at approximately 2 × 10⁴ cells/100 μl/well at 37 °C under 5% CO₂ for 24 h. One hundred microliters of 2 μM recombinant Igl, PBST or medium containing 200 units/ml penicillin G and 200 μg/ml streptomycin (Wako, Japan) was added and the cells were incubated for an additional 12 or 24 h under the same conditions. The cells were trypsinized and harvested after 0, 12 or 24 h of incubation and the number of cells per well was counted. The results are shown as the mean of 5 experiments with SD.