Ara c

Spinal neurons were obtained from E16 mouse embryos with a modified protocol from Jiao et al. (2020) (link). Briefly, the neonatal pups of C57BL/6 mice were anesthetized by isoflurane, decapitated, and vertebral columns were removed. The spinal cord was isolated and cut into 1-mm³ pieces after gently peeling off the meninges. The tissue pieces were digested by 0.25% trypsin for 10 min at 37°C, and then the cells were resuspended in a serum-free medium, including Neurobasal, B27, and penicillin/streptomycin (50 U/mL). 2.5 × 10⁵ cells were seeded in a 24-well plate at 37°C with 5% CO₂, and supplemented with 10 μM AraC (Sigma-Aldrich, St Louis, MO, United States) after 48 h. On the next day, the medium was replaced with Neurobasal containing supplements, excluding AraC, and changed every three days. The cells were cultured for 10 days and then OGD treatment was performed on them. Briefly, the spinal cord neurons were incubated in DMEM and cultured in an incubator containing 95% N₂/5% CO₂ for 4 h. Then, the DMEM was replaced with a standard neuronal culture medium for 24 h. The cells cultured in a standard neuronal culture medium in the presence of ambient 5% CO₂ served as the control. After OGD treatment, the neurons were either immediately treated with entinostat or not. The vehicle group received an equal volume of PBS.

Neuronal cultures were prepared and maintained as described in Aakalu et al. (2001) (link). Briefly, the cortex from E17 mice or hippocampus from P1 rats were dissected, dissociated with papain (Sigma) and plated on poly-D-lysine coated dishes. Neurons were maintained at 37°C and 5% CO₂ in Neurobasal-A plus B27 and GlutaMAX, (Life Technologies). One day after plating neurons were incubated with 3 µM AraC (Sigma) for two days, then the AraC was removed by changing the media. Unless otherwise indicated, neurons were used for experiments at DIV 14–21. For the experiments with KO mice cortical neurons were used, hippocampal neurons where used for the rest of the experiments.

In order to remove contaminating myoblasts from the myotube cultures, differentiated myotubes were transferred from DM to GM with 10 μM arabinosylcytosine (AraC; Sigma) for 48 h. After treatment with AraC, the cells were washed in phosphate buffered saline (1X PBS) and transferred back into DM for a 24-h recovery period prior to further treatments.