Isolation and Culture of Mouse HSCs

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).