Ph 797804

U2OS cells (purchased from ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D5796) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, E6541L), 2 mM L-Glutamine (LabClinics, M11–004) and 100 μg/ml penicillin-streptomycin (LabClinics, P11-010). For the inhibition of p38α, we used 1 μM PH-797804 (Selleckchem, S2726) or 10 μM SB203580 (Axon MedChem); both compounds can also inhibit p38β, although PH-797804 has been reported to preferentially inhibit p38α^{44 (link)}. MK2 was inhibited using 10 μM MK-2 Inhibitor III (Calbiochem) or 10 μM PF 3644022 (Sigma, PZ0188), whereas MSK activity was inhibited using 5 μM SB 747651 (Axon MedChem), and MNK activity was inhibited using 10 μM CGP-57380 (Sigma). MitoQ (kind gift from M. Murphy, Cambridge, UK) was used at 0.5 μM. Cisplatin was used at 50 μM, and the caspase inhibitors Z-VAD(OMe)-FMK (SM Biochemicals LLC SMFMK001) and Q-VD-OPH (SM Biochemicals LLC SMPH001) at 50 μM. The inhibitors were dissolved in DMSO and the total concentration of DMSO in the culture medium did not exceed 1%.

The following compounds were added to cell medium of hippocampal neurons: Cholesterol oxidase (Choox; Calbiochem ref.: 228250; 10 IU/ml); K252a (Tocris ref.: 1683; 1 μM); SB203580 (Shelleckchem ref.: S1076; 20 μM); PH797804 (Axon Medchem ref.: 1837; 2 μM); H89 (Tocris ref.: 2910; 50 μM); Chelerythrine (Tocris ref.: 1330; 10 μM); XL-184 (Tocris ref.: 5422; 1 μM); BAPTA-AM (Invitrogen ref.: B-6769, 10 μM). Experiments with the antioxidants cocktail in cultured hippocampal neurons used: N-Acetyl-L-Cysteine (NAC, Sigma-Aldrich ref.: A7250, 5 mM) and L-Glutathione reduced (GSH, Sigma-Aldrich ref.: G4251, 5 mM). Hippocampal slices were treated with Cholesterol oxidase (Choox; Calbiochem ref.: 228250; 10 IU/ml) for Cholesterol removal. Experiments for Cholesterol addition conducted in hippocampal slices were performed at 25°C. Methyl-β-cyclodextrin-Cholesterol (MβCD-Ch) solution was prepared freshly at use concentration in ACSF, containing 30 μM Cholesterol Water-soluble (Sigma-Aldrich ref.: C4951) and 5 μM Cholesterol (Sigma-Aldrich ref.: C3045).