Trehalose tre

The substrates glucose (G1), maltose (G2), maltotriose (G3), sucrose (Suc) and trehalose (Tre) were purchased either from Carl Roth, Sigma Aldrich (St. Louis, MI, USA) or VWR (Radnor, PA, USA). Maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) were obtained from Megazyme (Bray, Wicklow, Ireland). Acarbose (Acb) was produced and kindly provided by Bayer AG (Leverkusen, Germany). The substrate acarbose 7-phosphate (Acb-7P) was prepared enzymatically from acarbose and ATP using AcbK [13 (link)]. The reaction mixture (500 µL) containing 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 20 mM NH₄Cl, 10 mM ATP and 5 µM AcbK was incubated at 30 °C for 6 h. The enzyme was removed by ultrafiltration using an Amicon Ultra centrifugal filter (3 kDa MWCO, Merck Millipore) and the product was dried in a speed vacuum concentrator (Eppendorf Concentrator 5301, Eppendorf SE, Hamburg, Germany). Afterwards, the product was dissolved in a specific amount of water and verified by LC-ESI-MS, using the method described below (Section 2.9).

GSCs were mechanically dissociated and plated at a density of 10 × 10⁴ six-well microtiter plates. After 16 h, GSCs were treated for 72 h and 96 h.
The following chemicals and drugs were used: 450 μM Temozolomide (TMZ, Cayman Chemical Inc., Ann Arbor, MI), 10 μM z-VAD-FMK (Enzo Life Sciences, Rome, Italy), 5 μM CA074 (Chemicon International, Inc), 100 mM Trehalose (TRE, Sigma-Aldrich), 5 μM Quinacrine (QN, Sigma-Aldrich), 30 μM hydroxychloroquine (HCQ, Sigma-Aldrich), 10 μM calpain inhibitor I, 100 μM Necrostatin-1 (Enzo Life Sciences) 20 μM pepstatin A (Sigma-Aldrich), 100 μM deferoxamine (DFO, Sigma-Aldrich), and 20 μM ferrostatin 1 (Sigma-Aldrich). Compounds were dissolved in DMSO or in serum-free medium. Samples treated with vehicle alone were considered as control.