In Vitro | Kinase Assay: Cytosolic phosphorylation of Akt: Hela cells are serum starved for 1 hr and treated with IGF (100ng/mL) or SC79 (4 μg/mL) for 30 minutes. Cells are lysed in Lysis buffer containing 250 mM Sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA supplemented with protease inhibitors. Cells are passed through 25G needle several times and kept on ice for 20 minutes. Total cell lysate is taken at this point. Cell lysates are centrifuged at 100,000g for 30 minutes. Supernatant is collected as the cytosolic fraction. Pellet is washed with lysis buffer and represents the membrane fraction. Total cell lysate, cytosolic and membrane fractions are resolved by SDS-PAGE and analyzed for phospho-Akt (S473), Total Akt, Tubulin (cytosolic marker) and Orai1 (membrane marker) by western blotting. Cell Assay: HsSultan or NB4 cells (2.5 × 105) are plated in a 24-well plate in 500 μL of phenol red-free RPMI medium supplemented with 10% FBS. After incubation for 24 hours, each compound (8 µg/mL) is added and cultured for overnight (16–20 h). Fifty microliters of MTT solution (5 mg/mL in PBS) are added to each well. Following 2 hrs incubation, the purple formazan crystals are dissolved by directly adding in 500 μL of isopropanol with 0.1 M HCl to each well. After clearing the cell debris by centrifugation, the absorbance is measured at a wavelength of 570 nm. SC79 suppresses PHAKTM-GFP plasma membrane translocation, and enhances phosphorylation of all three Akt isoforms in HEK293, HeLa, HL60, NB4, and HsSulton (B cells) cells. SC79 reduces neuronal excitotoxicity and prevents stroke-induced neuronal death. SC79 restores proliferation of BRAT1 knockdown cells, and reduces the production of superoxide in mitochondria of MitoSox positive cells. |
In Vivo | In the permanent focal cerebral ischemia mouse model, SC79 (0.04 mg/g, i.p.) inhibits the cytosolic activation of Akt, and recapitulates the primary cellular function of Akt signaling, resulting in augmented neuronal survival. |