Cytarabine ara c

To generate mouse cortical neurons, Cdkl5^+/– female and Cdkl5^+/Y male or Cdkl5^+/– female and Cdkl5^+/Y/GFP-LC3^+/+ male mice were mated and the brains from littermate offspring harvested on embryonic day 15 (E15). The cortex was dissected from the rest of the brain under a dissecting microscope. Cortical cells were dissociated in 2.5% Trypsin (Thermo Fisher Scientific) and DNase I (Sigma-Aldrich), and resuspended in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were then plated on Nunc Lab-Tek II 8-well glass chamber slides (Thermo Fisher Scientific), or 12-well dishes coated overnight with 0.1% polyethyleneimine from Sigma-Aldrich. After 2 hours, the medium was replaced with Neurobasal medium supplemented with B27 (Invitrogen), 2 mM glutamine (Thermo Fisher Scientific), and 5 μM cytarabine (AraC) (Sigma-Aldrich) for 24 hours to suppress growth of non-neuronal cells, at which point fresh medium without AraC was added to culture neurons for 7 days.

Cytarabine (Ara‐C) and danorubicin were purchased from Sigma‐Aldrich (St. Louis, MI, USA). PTL and 3‐(1H‐1,2,3‐triazol‐4‐yl) pyridine (3‐TYP) were from Selleckchem (Houston, TX, USA). All compounds, except for in vivo studies, were reconstituted in dimethlysulfoxide, stored at 100‐mmol/l stock concentrations at −80°C, and used at the indicated doses suggested by the vendor. Flow cytometry antibodies, Alexa Fluor 647 Rabbit Anti‐Active caspase 3, PE‐Cy7 Mouse Anti‐Human CD38, APC‐H7 Mouse Anti‐Human CD45, and APC Mouse Anti‐Human CD34 were purchased from BD Pharmingen (San Jose, CA, USA). Immunoblotting antibodies, cleaved caspase 9, MCL1, BCL2, BAD, BAX, acetylated lysine and SIRT3 were purchased from Cell Signalling Technology (Danvers, MA, USA). Acetylated SOD2 and SOD2 antibodies were purchased from Abcam. ATPA (51) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).