BMI1 inhibition improves lesion burden in cerebral cavernous malformations
BMI1 inhibition improves lesion burden in cerebral cavernous malformations
The isolation and culture of BMECs was performed as previously described in Calabria et al, from Cdh5(PAC)-Cre-ERT2/CCM3flox/flox p30 mice. After dissection under sterile conditions, the brains were collected in buffer A (BSA1%, NaCl 153mM, KCl 5.6mM, CaCl2 2.3mM, MgCl2 2.6mM, Hepes 15mM, to volume with MilliQ H2O). Thereafter, the meninges were removed by rolling the brains on autoclaved Whatman Filter (10x15 cm). Brains were minced and mechanically digested for 1h in a 1:1 solution composed by buffer A and Worthington Collagenase (0,75% in BufferA) through the use of the MACS Octodissociator, with the protocol 37C_ABDK. In order to stop Collagenase action, buffer A was added and the solution was centrifuged for 10 min at 1200rpm, 4oC and low brake (2). To remove the myelin, the pellet of cells was resuspended with BSA 25%, avoiding the formation of bubbles, and centrifuged for 20min at 2600rpm, 4oC and low brake (2). A gradient was derived after centrifuge: BSA and myelin ring were removed and the residual cells were further digested with collagenase/dispase (1mg/ml, Roche, prepared in BufferA) and DNase I (39 U/ml) in BufferA for 15min at 37oC. Finally, the cells were washed with BufferA and put in culture in complete medium at a density of 1.5 x 104 cells/ml in Collagen I (Corning, from rat tail) pre-coated wells. BMECs were selected with a 2-day treatment of 4mg/ml puromycin. BMECs were cultured in sterile condition on collagen I-coated wells' plates. Complete medium for freshly isolated cells was composed by: MCDB-131 (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 20% North America Fetal Bovine Serum (HyClone), glutamine 2mM (Microtech), penicillin/streptomycin 100units/L (Microtech), heparin 100mg/mL (from porcine intestinal mucosa; Sigma) and endothelial cells growth supplement 100mg/mL (E2759, Sigma-Aldrich). BMECs were grown until confluence before starting the treatment. Thereafter, PTC-209 (Bmi1-inhibitor) was added to the confluent monolayer. fBMECs were incubated with drugs for 72h. The final concentration used was 1uM PTC209. Drug was dissolved in DMSO at 1000x concentration in order to keep final concentration of DMSO below 1%. Control cells were treated with DMSO at the same amount of the drug. Total RNA was isolated using the RNeasy Mini Kits (Qiagen Inc., Santa Clarita, CA, USA) and 500 ng total RNA was reverse transcribed with random hexamers (High-Capacity cDNA Archive Kits; Applied Biosystems), following the manufacturer's instructions. For genome-wide analysis, mRNA libraries were prepared with 500ng RNA using Illumina® Stranded mRNA Prep, Ligation.