Doxycycline

Doxycycline-inducible expressing 293T cell lines were seeded into a six-well plate and supplemented with 50 ng/ml Doxycycline (Takara). 12 h post-induction, mitochondrial and cytosolic fractions were separated by differential centrifugation using the Cell Mitochondria Isolation Kit (Beyotime Biotechnology). In brief, induced cells were trypsinized, washed with cold PBS, and homogenized in homogenizing buffer. The homogenate was spun at 600g for 10 min, and mitochondria in the supernate were pelleted at 11,000g for 10 min, and then resuspended in mitochondria storage buffer. The homogenate without mitochondria was centrifuged at 12,000g for 10 min, and the supernate was labeled as the cytosolic fraction. Protein concentration was determined by the BCA Protein Assay Kit (Thermo Fisher Scientific). Both mitochondria and cytosolic fractions were subjected to Western blot. An equal relative amount of mitochondria and cytosolic protein was loaded to maintain the original mitochondria/cytosolic ratio within the transfected cell between different transfections.

The U87MG parental, U87MG EGFRvIII, U178MG tet-EGFRvIII cell lines, and transformed mouse Ink4a/Arf(−/−) astrocytes, Ink4a/Arf(−/−) EGFRvIII cells, Ink4a/Arf(−/−) EGFRvIII Gefitinib/Erlotinib resistant cells, and Ink4a/Arf(−/−) PDGF-β cells have been previously described [29 (link), 31 (link), 42 (link)]. U87MG parental H2B-GFP and U87MG EGFRvIII H2B-GFP cells were constructed by infecting U87MG parental and U87MG EGFRvIII cells with a H2B-GFP construct [43 (link)] generously provided by Dr. David Pellman (Dana Farber Cancer Institute, Boston). TMZ (AK Scientific, Mountain View, CA) and BI2536 (ChemieTek, Indianapolis, IN) were dissolved in DMSO (Sigma Aldrich, St. Louis, MO). Doxycycline (Clontech, Mountain View, CA) was dissolved in deionized water. Cells were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% Pen-Strep (Gibco) and 1% GlutaMax (Gibco) unless otherwise specified. Tetracycline free serum (Clontech, Mountain View, CA) was used for experiments involving Doxycycline.

Animal handling and use was in accordance with a protocol approved by the Animal Care and Use Committee of Albert Einstein College of Medicine and NIH guidelines. Up to five mice were housed per cage, and animals were maintained on a 14-h light: 10-h dark cycle with food and water available. Progeny of transgenic IMP1 mice have been back crossed more than four generations to C57BL/6J and were crossed with male transgenic mice expressing the PyMT oncogene under the control of MMTV LTR promoter, provided by Jiufeng Li and were bred in house. For experiments, transgenic IMP1 mice used in the study were heterozygous for IMP1 transgene, MMTVrtTA and the PYMT oncogene and were all aged matched. Control mice were litter mates that did not express the IMP1 transgene. Mice were treated with doxycycline at weaning, three weeks of age. For induction of IMP1, doxycycline (Clontech) was added to the water at 1 mg/mL in water bottles covered with aluminium foil to prevent direct exposure of doxycycline treated water to light. Doxycyline treated water was changed weekly until the tumors reached the maximum size allowed in the protocol approved by the Animal Care and Use Committee of Albert Einstein College of Medicine and NIH guidelines, after which mice were sacrificed, there were no obvious signs of dehydration in mice that were administered doxycycline.

Human excitatory neurons differentiated from pluripotent stem cells by over-expression of lineage-specific transcription factor-Neurogenin 2 (Ngn2) as described before^{36 (link)}. In summary, one day before conversion, we dissociated stem cells into single cells with Accutase (Innovative Cell Technologies) and seeded at ~ 40 K cells into one 24 well plate pre-coated with Matrigel (BD Biosciences) in a medium supplemented with Thiazovivin (5 µM) (STEMCELL Technologies) and doxycycline (2 mg/ml, Clontech). After 6 h, we infected the cells with lentivirus containing Ngn2, RTTA, and Cre recombinase or ΔCre (truncated form of Cre, which is not functional and is used as control). The next day, we replaced the medium with neuronal medium N2/DMEM/F12/NEAA (Invitrogen) containing doxycycline (2 mg/ml, Clontech). We kept the cells in this medium for five days, and on day six, we added ~ 10 K mouse glia cells into each 24 well and replaced the culture medium with a serum-containing medium. We analyzed the cultures approximately 3–5 weeks after induction. To generate homozygous knockout neurons, we infected the neurons with LV- Cre or ΔCre one day after induction of the Ngn2 transcription factor.