Recilisib (Ex-RAD)

Kinase experiment:

PI3-kinase assays are performed using exponentially growing HFL-1 or freshly harvested murine bone marrow cells that are treated with increasing concentrations of Recilisib Sodium for 2 hours and then irradiated with 10 Gy IR. These cells are then returned to the incubator for 2 to 24 hours and lysed in HEPES pH 7.5 lysis buffer. PI-3K is immunoprecipitated using an anti-PI3 Kinas polyclonal antibody for 2 hours at 4°C. Protein A/G PLUS-Agarose is incubated with immunoprecipitates for 8-16 hours at 4°C and the resulting immunoprecipitates washed with twice HEPES pH 7.5 lysis buffer and once with the kinase buffer (20 mM Tris pH 7.5, 1mM EGTA, 10mM MgCl2, 2 mM DTT, 0.01% NP-40). L-α-Phosphatidylinositol (12.5 mM) and ATP (10 µM) are added to the kinase buffer (60 µL per sample) and incubated at 30°C for 30 minutes. The reaction is stopped by addition of 100 µL of 1N HCl and extracted by addition of 200 µL CHCl3/CH3OH (1:1). The extracted samples are vortexed, centrifuged and the lower organic phases containing phospholipids are dried at 27°C for 2 hours. The dried samples are resuspended in 10 µL of PI-4-P standard (0.5 mL CHCl3, 0.5 mL CH3OH, 2.5 µL HCl) and spotted on TLC plates (VWR). The spotted plate is subjected to thin layer chromatography in CHCl3/CH3OH/NH4OH (40:40:15). The TLC plate is dried and subjected to autoradiography.

Cell experiment:

For cytotoxicity assays, cells (1.0×105 cells/mL HPGM) are treated with various concentrations of Recilisib Sodium or vehicle without radiation treatment for 2 or 24 hours. The cells are washed and plated into methocult using gridded dishes. The total number of colony forming units (CFUs) is determined 14 days post-plating by microscopic observation using an Olympus IMT-2 microscope.

References:

[1]. Kang AD, et al. ON01210.Na (Ex-RAD) mitigates radiation damage through activation of the AKT pathway. PLoS One. 2013;8(3):e58355.