Dulbecco s modified eagle s medium

The animal experiments were approved by the animal care and use committee of Vrije Universiteit Brussel (Permits 13-212-1 and 14-212-4) and the institution’s guidelines for the care and use of laboratory animals in research were strictly followed. The HSC isolation method for male BALB/c mice (aged 15–20 weeks) was performed as previously described^{18 (link)}. Total c-Jun N-terminal kinase (JNK) mice were provided by Professor Christian Trautwein and were originally described in Zhao et al.^{19 (link)} and Cubero et al.^{20 (link)}. After isolation, a fraction of the cells was collected for RNA or cytospinned and the rest was cultured in Dulbecco’s modified Eagle’s medium (Lonza, Braine-l’Alleud, Belgium) supplemented with 10% fetal bovine serum (Lonza) at 37 °C in a humidified atmosphere with 5% CO₂. Treatment with 5 and 10 µM JNK V inhibitor (Calbiochem, Merck, Overijse, Belgium) was started from the moment of seeding and refreshed every day. VG-1 (Gift from Prof. Coldham) and NMDi-14 (R&D systems) treatments were initiated from the moment of seeding. Solvent controls were treated with 0.1% dimethyl sulfoxide. Bright-field images were taken with an Axioskop light microscope (Carl Zeiss, Zaventem, Belgium).

HeLa cells were obtained from the American Tissue Culture Collection. Cells were grown in Dulbecco’s modified Eagle’s medium (Lonza) supplemented with 10% (v/v) of fetal-calf serum (Lonza). Cells were maintained at 37 °C in a humidified atmosphere containing 5% (v/v) CO₂.

HuH-7 cells were cultured in Dulbecco's modified Eagle's medium (Lonza, Cologne, Germany) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 4 mM L-glutamine (Lonza) and 100 U/ml penicillin/streptomycin (Biochrom KG, Berlin, Germany) in a humidified incubator at 37 °C and 5% CO₂ atmosphere. For treatment, 10 ng/ml recombinant human cytokine IL-6 or 5 ng/ml TGF-β (both Peprotech, Hamburg, Germany) were used. Cell lines are tested regularly for mycoplasma contamination in the laboratory.

The HeLa cells were grown in Dulbecco's modified Eagle's medium (Lonza), and the SK-N-BE(2)C cells were maintained in RPMI 1640 without l-glutamine (Lonza). Each medium was supplemented with 10% fetal calf serum (PAA Laboratories), 100 units/ml penicillin (Lonza), 100 μg/ml streptomycin (Lonza), and 2 mml-glutamine (Lonza).
The cells were transiently transfected or co-transfected by means of lipofection (FUGENE HD, Promega) as described previously (21 (link), 25 (link), 26 (link)) using 1.5 × 10⁵ SK-N-BE(2)C or 5 × 10⁴ HeLa cells.
In mammalian two-hybrid experiments, 80 fmol of each expression vector were combined with 160 fmol of pG5LUC plasmid. The luciferase assay was carried out using the Dual-Luciferase reporter assay system (Promega) as described previously (26 (link), 27 (link)). All of the transfections were performed in triplicate, and each construct was tested in at least three independent experiments using different batches of plasmid preparation. The numbers of independent transfection experiments are indicated in the figure legends.

Vero cells expressing human CD150 (Vero-hCD150) were a kind gift from Prof Yusuke Yanagi, Kyushu University, Japan. The cells were grown in Dulbecco’s modified Eagle’s medium (Lonza) supplemented with 10% FBS, penicillin, streptomycin and l-glutamine (D10F medium)^{40 (link)}. A human B-lymphoblastoid cell line (BLCL) was cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin, streptomycin and l-glutamine (R10F medium)^{41 (link)}. The cell line was generated in-house by Epstein-Barr virus-transformation of PBMC of a healthy adult donor. Human melanoma Mel-JuSo cells transfected with the full-length MV-Edmonston fusion (F) or hemagglutinin (H) genes (Mel-JuSo-F or Mel-JuSo-H)^{42 (link)} were grown in R10F medium. Virus isolations were performed by inoculation of Vero-hCD150 cells or BLCL with PBMC or virus transport medium from swab samples in two-fold serial dilutions (eight replicates per dilution). Viral cytopathic effects were monitored over a period of 3–7 days and a virus titre was calculated by determining the 50% endpoint using the formula of Reed and Muench⁴³ . One of the virus isolates (MVi/Dodewaard.NLD/29.13; genotype D8) was used for full genome sequencing as described previously (GenBank accession number: MG912592)^{44 (link)}. This isolate is available via the European Virus Archive [https://www.european-virus-archive.com].

Human Embryonic Kidney 293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (Lonza) supplemented with 10% Fetal Calf Serum (Sigma) and penicillin-streptomycin (Life Technologies). Transfection in 6 well plates was performed for 48 hours using 2.0 ug of plasmid and Lipofectamine 2000 (Life Technologies) following the manufacturer’s guidelines.