Recombinant human mouse wnt 5a

Recombinant Human/Mouse WNT-5A and Recombinant Human/Mouse WNT-5B were obtained from R&D Systems Inc. (Minneapolis, MN, USA). Rabbit anti-Prosurfactant Protein C (proSP-C) was purchased from EMD Millipore Corporation (Amsterdam-Zuidoost, North Holland, The Netherlands). Mouse anti-acetylated α tubulin was purchased from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Mouse anti E-cadherin was purchased from BD Biosciences (Bedford, MA, USA). Matrigel and cell culture inserts were obtained from Corning Incorporated (New York, NY, USA). CD31, CD45, and CD326 (EpCAM) microbeads were purchased from Miltenyi Biotec (Leiden, The Netherlands).

Recombinant Human/Mouse WNT5A was purchased from R&D Systems (Minneapolis, US), diluted in PBS with 1% BSA. For experiments with recombinant WNT5A, HD-LECs were pre-treated for three days with 10 µM LGK974 (Selleck Chemicals). We used the following inhibitors: BIO (2 µM, Sigma), EHT 1864 (10 µM, Tocris Bioscience, Bristol, GB), Fasudil (10 µM, Selleck Chemicals), FH535 (10 µM, Tocris Bioscience), IM-12 (10 µM, Selleck Chemicals), JNK-IN-8 (5 µM, Merck Millipore), NSC23766 (100 µM, Selleck Chemicals), SP600125 (10 µM, Santa Cruz Biotechnology), Y-27632 (50 µM, Selleck Chemicals). For all inhibitors DMSO (Sigma) was used as solvent.

HL-1 mouse atrial cardiomyocytes were a kind gift from Dr. Pasi Tavi (University of Eastern Finland). HL-1 cells were maintained in Claycomb medium (Sigma) supplemented with 10% FBS, 0.1 mM norepinephrine, 50 U/ml penicillin, 50 U/ml streptomycin, and 2 mM UltraGlutamine (Lonza). Culture plates were coated at 37 °C with a solution containing 0.02% gelatin and 10 μg/ml fibronectin. Seeding densities were 140,000 cells/6-well plate well, 100,000 cells/12-well plate well, and 5,000 cells/96-well plate well. H9c2 rat cardiomyoblasts were purchased from ATCC and maintained in DMEM with 1.5 g/l NaHCO₃, supplemented with 10% FBS, 50 U/ml penicillin, 50 U/ml streptomycin, and 2 mM UltraGlutamine. Seeding density was 100,000 cells/6-well plate well. Cells were routinely checked for mycoplasma infection using MycoAlert Mycoplasma Detection Kit (Lonza). For hypoxia-reoxygenation experiments, the cells were cultured in 1% O₂ in a hypoxic work station (InVivo₂, Ruskinn Technology Ltd.) for the indicated periods of time, and returned to normal cell incubator (21% O₂) for reoxygenation. In ROR1 ligand activation experiments, 200–400 ng/ml of recombinant human/mouse Wnt-5a (R&D Systems) was added to medium at the time of plating (for MTT assays) or 24 h after plating for 30–60 min (for Western analyses).