Ethylene glycol tetraacetic acid (egta)

PASMCs were identified by expression of α-actin (≥ 95% of the cells) by immunofluorescence using an antibody against α-SMA (1:200, Abcam, USA). Primary human pulmonary artery SMCs (ScienCell Research Laboratories, USA) were maintained in a Petri dish containing prechilled PBS, 2% penicillin–streptomycin, Ca²⁺-free HBSS, and 0.5% fetal calf serum mixed with 20% DMEM/F12 medium (Billups-Rothenberg, Del Mar, USA). Then, the cells were cultured in an atmosphere containing 5% CO₂ and 21% O₂ or 3% O₂ in a humidified incubator at 37 °C for 3, 6, 12, 24, and 48 h for normoxia or hypoxia treatments, respectively. For cell proliferation, Ca²⁺ imaging, and detection of the phosphorylation signaling pathway, PASMCs were pretreated with cyclopiazonic acid (CPA), nifedipine (Nifed), EGTA (Santa Cruz, USA), BAPTA/AM (Santa Cruz, USA), LY294002 (Santa Cruz, USA), Perifosine (Santa Cruz, USA), LY317615 (Santa Cruz, USA), rapamycin (Santa Cruz, USA), or PD98059 (Santa Cruz, USA) and seeded onto 25 mm glass coverslips as indicated. Then, various concentrations of recombinant human RELM-β protein (0–40 ng/ml, Abgent, USA) were used to stimulate PASMCs for various periods (0–72 h). Cells of passages 3–7 were used in the experiments. When cells in the logarithmic growth phase reached a density of 70%, cell cycle was synchronized by incubation in the serum-free medium for 24 h.

Cytoskeleton (CSK) buffer was used for cell lysis and sample preparation and consisted of 100 mM NaCl, 300 mM sucrose, 10 mM Tris pH 7.5, 3 mM MgCl₂, and 1mM EGTA (Santa Cruz Biotechnology, Heidelberg, Germany, #Sc-3593A). The buffer for cell disruption and nuclei isolation was prepared from the CSK buffer and contained 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA, #T9284), 1.2 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, #000000010837091001), and 1×protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA, #P8340-5ML).