Hepes

During the microinjection and up to the electrophysiological experiment the oocytes were kept in a solution containing 88 mM NaCl, 1 mM KCl, 2.5 mM NaHCO₃, 10 mM HEPES (AppliChem GmbH, Darmstadt, Germany), 0.82 MgSO₄, 0.33 mM Ca(NO₃)₂, 0.33 mM CaCl₂, 100 U/mL penicillin (GIBCO, ThermoFisher Scientific) and 100 µg/mL streptomycine (GIBCO, ThermoFisher Scientific). The pH was adjusted to 7.5. During the experiment the voltage-clamped oocyte was constantly superfused by a solution containing 90 mM NaCl, 1 mM KCl, 5 mM HEPES (AppliChem), 1 mM MgCl₂ and 1 mM CaCl₂ (pH 7.4). GABA and test compounds were added to this solution. GABA concentrations were chosen according to previously determined concentration–response relations for the different human GABA_A receptor subunit combinations. Concentrations evoking approximately 10% of a maximal response were selected, i.e., 8 µM for α1β2γ2, 6 µM for α2β3γ2, 10 µM for α3β3γ2 and 5 µM for α5β3γ2. Solutions containing GABA or test compounds were freshly prepared at least every second day. Stock solutions of basmisanil, flumazenil and β-CCM were prepared in DMSO at 10 mM concentration. Compounds were further diluted in DMSO and finally in extracellular saline to reach a DMSO concentration of 0.1%. The same amount of DMSO was added to the control solutions without test compounds.

U251 and GL261 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% fetal bovine serum (FBS) (Gibco) supplemented with 100 U/ml penicillin G (Sigma-Aldrich), 100 μg/ml streptomycin sulfate (Sigma-Aldrich), 6mM hepes (Applichem), 1.6 mM L-glutamine (Sigma-Aldrich), 50 μM β-mercaptoethanol (Biorad). U251 and GL261 harvested using accutase (Invitrogen) according to manufacturer’s procedure. To obtained hNS, U251 were dedifferentiated and maintained in neurosphere medium (DMEM/F12) medium supplemented with, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, 6mM hepes, 1.6 mM L-glutamine, 50 μM β-mercaptoethanol, B-27 supplement (Invitrogen), 20 ng/mL recombinant human basic fibroblast growth factor (basic-FGF) (Invitrogen,) and 20 ng/ml recombinant human epidermal growth factor (EGF) (Invitrogen) and 50 U/ml heparin (Sigma). To obtained mNS, GL261 cells were dedifferentiated in the same medium using mouse basic FGF and mouse EGF. For each passage, spheres were dissociated with accutase (Invitrogen) and passaged into fresh media. YT-Indy NK cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate 6 mM hepes free acid, 1.6 mM L-glutamine, and 50 μM β-mercaptoethanol.

The Ewing sarcoma cell line A673 was a kind gift of Dr. S. Lessnick (Nationwide Children’s Hospital, Columbus, OH; Feb 2014) and contained (for prospective synthetic lethal screens) a retroviral pSRP shRNA expression vector with control insert [33 (link)]. The human embryonic kidney cell line HEK293 (ACC 305; DSMZ, Braunschweig, Germany) was from our institutional repository. Both cell lines were authenticated by short tandem repeat examination after pooled screens to confirm cell line identity in screen hits. The HEK293T packaging cell line was purchased from Thermo Fisher Scientific (Cat-No. HCL4517; Waltham, MA; Sept 2012). All cell lines were cultured at 37°C with 5% CO₂ in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) and for HEK293T with 20 mM HEPES (AppliChem, Darmstadt, Germany). All cell lines were regularly tested to be free of mycoplasma.