Bapta am

Polyclonal rabbit anti-focal adhesion kinase (FAK) and polyclonal rabbit anti-phospho-Tyr397-FAK were purchased from Cell Signaling (Danvers, MA, United States), polyclonal rabbit anti-MMP1 from Abcam (Cambridge, United Kingdom) and monoclonal anti-β-actin from Sigma (Munich, Germany). Polyclonal rabbit anti-mouse, anti-goat and anti-rabbit IgGs were acquired from Dako (Glostrup, Denmark). Apigenin and luteolin were purchased from Sigma (Munich, Germany) and human recombinant MMP1 from Sigma-Aldrich (SRP3117, St. Louis, MO, United States). U73122 was obtained from Calbiochem (Darmstadt, Germany), BAPTA-AM was from Santa Cruz Biotechnology (Heidelberg, Germany).
siRNAs targeting human MLC2 (MYL2; SMART pool, ON-TARGET PLUS, Cat. No.: L-011087000005, siRNAs targeting human FAK (SMART pool, ON-TARGET PLUS, Cat. No.: L-003164000005) were ordered from Dharmacon (Gene Expression and Gene Editing, GE Healthcare, Lafayette, CO, United States). Non-targeting (n.t.) control siRNA (Silencer^® Select Negative Control No. 1 siRNA, Cat. No.: 4390843) was purchased from Ambion (Life Technologies, Carlsbad, CA, United States). All siRNAs were re-suspended in RNAse-free water to yield a stock concentration of 20 μM.

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.

MG63 (osteosarcoma) and sw-1353 (chondrosarcoma) cell lines were purchased from the American Type Culture Collection (Teddington, UK) and cultured in DMEM (Sigma- Aldrich, St Louis, MO, USA) medium supplemented with 10% v/v Fetal Bovine Serum (FBS; Gibco, Invitrogen Life Technologies, Barcelona, Spain), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Spain) in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were sub-cultured once a week using a 0.25% trypsin solution. Compound concentrations used for the experiments were: 1 mM melatonin, 5 µM BAPTA, 3 mM calcium chloride, 100 µM trolox and 100 µM ascorbate (Sigma-Aldrich, St Louis, MO, USA). Culture flasks and dishes were obtained from Fisher Scientific (Madrid, Spain). Fluo-3AM and Rhod-2 were purchased from Invitrogen (Invitrogen Life Technologies, Barcelona, Spain) and BAPTA/AM was acquired from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA). All other reagents were purchased from Sigma (Sigma-Aldrich, St Louis, MO, USA), unless otherwise indicated.

RNA was extracted using RNeasy Mini Kit (Qiagen, USA) from RASFs untreated (control), treated with celastrol [1 μM], or celastrol in the presence of BAPTA/AM [10 μM] (Santa Cruz, USA) for 24 h. RNA concentration was determined using the NanoDrop 2000c Spectrophotometer (Thermo Scientific) and 1 μg of RNA was used to synthesize cDNA with RT2 First Strand Kit (Qiagen, USA). Three independent biological samples were employed.

The colon cancer cell lines were transferred onto six-well plates and maintained with different doses of polydatin (0, 100, 200, and 300 μM), or polydatin (300 μM) with calcium chelators, including 2-aminoethoxydiphenyl borate (2-APB; Cat No: D9754, Sigma-Aldrich), and 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid tetrakis (BAPTA/AM; Cat No: sc-202488, Santa Cruz Biotechnology), for 24 h. These cells were stained by Rhod-2 dye for 30 min. Fluorescence levels were estimated using a flow cytometer. The experiment was conducted in triplicate.