As biotin

Cells were treated with an arsenic-biotin conjugate (As-Biotin) (CAS Number: 212391-23-6, Toronto Research Chemicals, Inc) at a final concentration of 20 μM for 2 hrs. This compound has been previous used to isolated arsenic binding proteins as it will bind to thiol groups (6 (link), 8 (link), 37 (link), 38 (link)). After lysis in phospho-lysis buffer, 500 μg of protein was diluted in buffer and the arsenic-protein complexes were then isolated with streptavidin agarose beads by tumbling at 4°C overnight (Invitrogen Life Technologies). For recombinant protein, purified AMPKα1 subunit (Novus Biologicals) or the active holoenzyme containing AMPKα1/β1/γ1 (SignalChem) protein was incubated for 30 minutes with As-Biotin at 30°C. The arsenic-protein complex was then isolated with streptavidin agarose beads at 4°C for 1 hr (Invitrogen Life Technologies, Carlsbad, CA). The beads were washed 3 times with phospho-lysis buffer not containing Triton-X 100. The samples were then boiled for 5 min at 95 °C in 2x Laemmli sample buffer and subject to SDS-PAGE. Immunoblotting was performed as described above.

Established GBM cell lines (U87, LN229, and LN18) were grown in DMEM (Thermo Fisher) with 10% FBS (Thermo Fisher) and gentamycin (1 mg/mL). The identity of established cell lines was authenticated by short-tandem repeat (STR) analysis (Genetica DNA Laboratories) in August 2016. PDX cell lines were grown in serum-free Neurobasal medium (Thermo Fisher) with N-2 Supplement at a 1X final concentration (Thermo Fisher), EGF (50 ng/mL), bFGF (25 ng/mL), and penicillin/streptomycin (10 U/mL). Patient-derived MES (83Mes) and PN (AC17PN) GSC lines have been previously described (2 (link), 21 (link)). GSCs were grown in DMEM/F12 with EGF (20 ng/mL), bFGF (20 ng/mL), heparin (5 μg/mL), B27 (2%), and gentamycin (1 mg/mL) in nonadherent flasks. All cells were grown at 37 C in 5% CO₂. Pharmaceutical-grade ATO (Trisenox) used for in vitro and in vivo experiments was purchased from Teva Pharmaceuticals. The MNK inhibitor, CGP57380, was purchased from Santa Cruz Biotechnology. The arsenic–biotin conjugate (As-Biotin) was purchased from Toronto Research Chemicals and has been previously described (23 (link)).