Tert butyl hydroperoxide

To induce NF-κB signalling, human recombinant TNFα (PeproTech) was used at 10 ng/ml for 0.5-6 h. ATM inhibitor AZD1390 (SelleckChem) was used at a final concentration of 10 nM for at least 1 h and up to 9 days (DMSO served as a control) in HMC3 cells and at 100 nM in LUHMES cells during days 2–5 of differentiation. For long-term inhibitor treatments, the medium with inhibitor was renewed every 48 h. For the inhibitor wash-out, cells were washed with PBS and cultured in the absence of inhibitor for an additional 2 days. To induce ATM-dependent phosphorylation, cells were treated with 1 μM camptothecin (CPT; Cambridge Bioscience) for 1 h. To induce apoptosis, cells were treated with 0.33 μM staurosporine (Merck) for 16 h. Treatment with 2 μM hydrogen peroxide for 5 min (H₂O₂; Merck) was used to induce DNA strand-breaks and oxidative damage. To induce persistent DNA damage, cells were treated with 0.5 μM etoposide, or DMSO as a control, or with 100–250 μM tert-butyl hydroperoxide (tBHP) for up to 6 days (Merck). The medium with tBHP or etoposide was renewed every 24 or 48 h, respectively. A scavenger of superoxide radicals, Tiron (Merck), was used at 10 μM over a 72-h period, and the medium containing the compound was changed daily.

All the chemicals, including Folin-Ciocalteu’s phenol reagent, polyvinylpyrrolidone (PVP), sodium carbonate, tannic acid (98% purity), aluminum chloride hexahydrate (AlCl₃·6H₂O; Ph Eur purity), quercetin (98% purity), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH; 95% purity), 2,2-azobis (2-methylpropionamidine) dihydrochloride (AAPH; 97% purity), 2,2-azino-bis (3-thylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; 98% purity), trolox (97% purity), ferrozine (97% purity), iron (II) sulfate heptahydrate (FeSO₄·7H₂O; 99% purity), iron (III) chloride (FeCl₃·6H₂O; 97% purity), hydroxylamine hydrochloride (98% purity), rutin (99% purity), bovine serum albumin, glucose, fructose, sodium azide, iron (II) chloride (FeCl₂·4H₂O; 99% purity), tert-butyl hydroperoxide (tBOOH; 80% purity), and methanol were purchased from Merck (Darmstadt, Germany). RPMI 1640 medium and fetal bovine serum were provided by Aurogene (Rome, Italy).