MK 2206

Neonatal rat cardiomyocytes (NRCMs) were isolated from 1- to 3-day-old Sprague-Dawley (SD) rats and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning) containing 4.5 g/L glucose supplemented with 10% horse serum (Gibco) and 5% fetal bovine serum (FBS, BioInd, Israel) as described before [20 (link), 21 (link)]. To induce apoptosis, cardiomyocytes were treated with oxygen glucose deprivation/reperfusion (OGDR). Briefly, cardiomyocytes were first cultured for 8 h with serum-free no glucose DMEM (Gibco) in an air-tight chamber with a humidified hypoxic atmosphere containing 5% CO₂ and 95% N₂ at 37 °C. After exposure to oxygen glucose deprivation for 8 h, the culture medium was replaced with serum and glucose-containing DMEM and transferred to a normal incubator for recovery for 12 h.
To study the role of CRAMP in OGDR-induced apoptosis, NRCMs were simultaneously treated with the rat CRAMP (rCRAMP) bought from Innovagen AB (Lund, Sweden) at a dose of 0.1 mg/L for 48 h, or transfected with the siRNA targeting rCRAMP (100 nM, Ribobio, Guangzhou) for 48 h. To study whether protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) activation contributed to the role of CRAMP in OGDR-induced apoptosis, NRCMs were treated with the rCRAMP (0.1 mg/L, 48 h) in the presence or absence of Akt inhibitor MK2206 (10 nM, 24 h, Selleck) or MEK inhibitor PD98059 (50 μM, 24 h, Selleck). To clarify the upstream regulators of CRAMP, NRCMs were transfected with c-Jun or Rela siRNAs (100 nM, Ribobio, Guangzhou) in the presence or absence of rCRAMP siRNA for 48 h. In all in vitro experiments, the OGDR-induced apoptosis (8 h deprivation/12 h reperfusion) was conducted in the last 20 h of cell treatment.

The TMZ-resistant glioma cell lines, T98G-R and U87-R, were derived from the parental cell lines (T98G and U87) by treatment with gradually increasing concentrations of TMZ. Human malignant glioma cell lines T98G, U87, U251, U343, MGR2, and Hs683 were cultured in high-glucose DMEM medium (Glibco, United States), supplemented with 10% (v/v) fetal bovine serum (HyClone, United States), 1% penicillin and streptomycin. All cell lines were grown in a humidified incubator at 37°C with 5% CO₂.
Temozolomide and epidermal growth factor (EGF) were purchased from Sigma–Aldrich Corporation Chemicals. A939572 and LY294002 were purchased from MedChem Express. MK2206 was purchased from Selleck Chemicals. All reagents above were dissolved in dimethylsulfoxide (DMSO) (Sigma). For the Akt activation, cells were starved by serum-free medium incubation for 24 h, and then treated with 30 ng/mL EGF for 30 min. The exposed concentrations of TMZ, MK2206, and LY294002 were 200, 5, and 20 μM, respectively.

Primary antibodies used for Western blotting: anti-Akt-1 (1:1000 dilution, cat.# 4691, Cell Signaling); anti-phospho-Akt-1 (S473) (1:1000 dilution, cat.# 9018, Cell Signaling); anti-phospho-Akt-1 (T308) (1:1000 dilution, cat. 13038, Cell Signaling); anti-β-tubulin (1:1000 dilution, cat. 2128, Cell Signaling); anti-ARID1A (1:1000 dilution, cat.# NB100-55334, from Novus Biologicals). PARPi were purchased: ABT-888 from Enzo Life Sciences (cat.# ALX-270-444-M001); Olaparib from Selleck Chemical LLC (cat.# S1060). An inhibitor of PI3K LY294002 was obtained from Selleck Chemical LLC (cat.# S1105) and allosteric Akt inhibitor MK-2206 from Cayman Chemical (cat.# 11593). ARID1A Silencer™ siRNA and Silencer™ Select Negative siRNA Control were purchased from ThermoFisher Scientific (cat.# 4392420 and 4390843). Lipofectamine RNAiMAX (cat.# 13778075, ThermoFisher Scientific) was used for siRNA transfection according to manufacturer recommendations.