Panobinostat

All reagents were of analytical grade. Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), non-essential amino acid (NEAA), and MEM vitamin solutions were obtained from GIBCO Life Technologies (Grand Island, NY, USA). Penicillin, streptomycin, sodium pyruvate, PTU, tricaine, bovine serum albumin (BSA), diaminobenzydine (DAB), and mouse anti-mouse vimentin antibody (Vim 13.2 clone) were from Sigma-Aldrich (St. Louis, MO, USA). Paclitaxel, panobinostat, and everolimus were from MedChemExpress (Monmouth Junction, NJ, USA). The Annexin-V/propidium iodide double staining kit was from Immunostep Biotec (Salamanca, Spain). The ONE-Glo™ Luciferase Assay System was from Promega (Milan, Italy). Rat anti-mouse Ki-67 antibody (TEC-3) was from Dako (Santa Clara, CA, USA). Rabbit anti-human cleaved caspase 3 (Asp175) was from Cell Signaling (Danvers, MA, USA). Biotinylated anti-mouse IgM, anti-rat, and rabbit antibodies were from Abcam (Cambridge, UK). Biotin Avidin system Vectastain ABC reagent was from Vector Laboratories (Burlingame, CA, USA).

SNDX-5613, SNDX-50469, ziftomenib (KO-539), ATRA, SY-1425, Cytarabine, Daunorubicin, Selinexor (KPT-330), Entinostat, Panobinostat, and Adavosertib were obtained from MedChem Express (Monmouth Junction, NJ). Cycloheximide was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX). All compounds were prepared as 10 mM stocks in 100% DMSO and frozen at −80 °C in 5–10 µL aliquots to allow for single use, thus avoiding multiple freeze-thaw cycles that could result in compound decomposition and loss of activity. For in vivo studies, SNDX-5613 was obtained from Syndax Pharmaceuticals under an MTA and reconstituted per the manufacturer’s instructions.

EJ and HCT116 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM), while H522 cells were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI) (Thermo Fisher). Media were supplemented with penicillin-streptomycin (50 units/ml) and 10% Foetal Bovine Serum (FBS). EJp53 and EJp21 cells were cultured in complete culture media supplemented with 750 μg/ml geneticin and 100 μg/ml hygromycin, while EJp16 cells were maintained in media supplemented with 2 μg/ml puromycin and 100μg/ml hygromycin. To maintain EJ cells proliferating, 1 μM tetracycline was added to the culture media. To induce senescence, cells were trypsinized, washed with 1x phosphate buffered saline (PBS), and centrifuged at 244 g for three minutes. This step was repeated three times. Cells were cultured for a further 6 days in the absence of tetracycline to ensure the establishment of senescence. HCT116 cells were treated with 0.2 μM doxorubicin for 3 to 4 days to induce senescence. H522 cells were irradiated at 8Gy and cultured for 6 days to induce senescence. The drugs used were: CUDC-907 (APExBIO, #A4097), Dactolisib (Selleckchem, #BEZ235), Panobinostat (MedChemExpress, #404950-80), Buparlisib (APExBIO, #BKM120), CI-994 and Rocilinostat (Adooq Bioscience, #ACY-1215).

Alisertib (MedChemExpress, Monmouth Junction, NJ), bortezomib (MedChemExpress), panobinostat (MedChemExpress), ponatinib (MedChemExpress), MSN1-Leu (synthesized in our lab, [20 (link)]) were sterilely prepared and dissolved in 0.5% DMSO and sterile PBS. Bevacizumab (SelleckChem) is water soluble, and was directly dissolved in sterile PBS. Drugs were administered by pipetting 15μL of drug solution directly on top of (but not contacting) the CAM tumor on embryonic development day 11, 13, 15. Each experiment contained 1–5 biological replicates per treatment group (at time of ultrasound) and there were at least three experimental replicates for each drug (n = 3–24 per drug treatment). Vehicle treatments (0.5% DMSO in sterile PBS) from all drug experimental replicates were combined to form one large vehicle group for further analysis (DIPGXIIIp* n = 38, DIPGIV n = 19).