Calyculin a

U87 cell line was purchased from ATCC and maintained in DMEM (Life Technologies), supplemented with 10% FBS (Sigma-Aldrich), and 1% antibiotic-antimycotic (Life Technologies). The 4 primary glioblastoma stem cell lines (OSU-2, −11, −20, and −53) were isolated from glioblastoma patient tissues and authenticated by a neuropathologist at The Ohio State University. GSCs were maintained in DMEM:F12 (Life Technologies) supplemented with B27 supplement (Gibco), recombinant human FGF (Gibco), recombinant human EGF (Gibco), 10% FBS and 1% antibiotic-antimycotic (Life Technologies). Normal human astrocytes were obtained from Lonza and maintained in Clonetics AGM Astrocyte Growth Medium (Lonza) supplemented with 10% FBS and 1% antibiotic-antimycotic (Life Technologies). All cells were cultured at 37°C under a gas phase of 95% air and 5% CO₂. For calyculin A treatment, cells were treated with 100 nM calyculin A (Santa Cruz) for 30 minutes, and then harvested for western blot. For combination calyculin A and CX-4945 treatment, cells were treated with 10 μM CX-4945 (Selleckchem) for 24 hours, followed by 100 nM calyculin A for 30 minutes.