Sodium valproate

Pazopanib and AR42 and other kinase inhibitors were purchased from Selleckchem (Houston, TX). Sodium valproate was from Sigma (St. Louis, MO). Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). J28, T24, MEL24, MEL28, OVCAR, PAI, SKOV3, HCC38, SUM149, A549, H460 and H1975 cells were purchased from the ATCC and were not further validated beyond that claimed by ATCC. Cells were re-purchased every ∼6 months. Characterized PDX melanoma isolates expressing mutated active B-RAF were from the University of Pittsburgh's melanoma cell bank. H&N PDX tumor isolates were kindly provided by Dr. Lee. ADOR cells were a gift to the Dent lab from a female NSCLC patient. Spiky, CTG-1703, CTG-1677 PDX ovarian cancer cells were provided by Dr. Karen Paz (Champions Oncology, NJ). Commercially available validated short hairpin RNA molecules to knock down RNA/protein levels were from Qiagen (Valencia, CA) (Supplementary Figures 24 and 25). Reagents and performance of experimental procedures were described in refs: [1 (link)–8 (link)].

For experiments with zebrafish larvae, dry samples and compounds were dissolved in 100% dimethyl sulfoxide (DMSO, spectroscopy grade, Acros Organics (Geel, Belgium)) as 100-fold concentrated stocks and diluted in embryo medium to a final concentration of 1% DMSO content, except for PTZ and valproate which were dissolved in embryo medium (0% DMSO). Of note, for zebrafish exposure to valproate the DMSO content of the embryo medium was adjusted to 1% DMSO in line with other treatments. Control groups were treated with 1% DMSO (VHC) in accordance with the final solvent concentration of tested samples or compounds. For mice experiments, a mixture of polyethylene glycol M.W. 200 (PEG200, >95% purity, Acros Organics (Geel, Belgium)) and 100% DMSO (1:1 PEG200:DMSO) was used as solvent and VHC. Pentylenetetrazole (≥99% purity) and valproate (sodium valproate, ≥98% purity) were purchased from Sigma-Aldrich (Overijse, Belgium).

The human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (ATCC). Gibco media RPMI 1640 and fetal bovine serum (FBS) were used (Invitrogen Co., Carlsbad, CA, USA). Anti-caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, caspase-8, cleaved caspase-8, and p21Waf/cip1 [below as p21) antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-cyclin A, cyclin D1, cyclin E, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Cambridge, UK). Sodium valproate, Cell Counting Kit-8 (CCK-8), cell apoptosis related reagents, cell cycle detection reagents, and all other reagents used in the present study were all Sigma products (St. Louis, MO, USA).

Sodium valproate was from Sigma (St. Louis, MO). Neratinib was supplied by Puma Biotechnology Inc. (Los Angeles, CA). Sorafenib tosylate, dasatinib, ruxolitinib, dabrafenib, trametinib and sildenafil were from Selleckchem (Houston TX). Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). All “H” series NSCLC lines were purchased from the ATCC and were not further validated beyond that claimed by ATCC. Cells were re-purchased every ~6 months. ADOR cells were a gift to the Dent lab from a female NSCLC patient. Spiky ovarian cancer cells were kindly provided by Dr. Karen Paz (Champions Oncology, NJ). Commercially available validated short hairpin RNA molecules to knock down RNA / protein levels were from Qiagen (Valencia, CA) (Supplementary Figure 24). Control IgG, anti-PD-1 and anti-CTLA4 endotoxin-free antibodies were purchased from Bio-X cell (West Lebanon, NH). Reagents and performance of experimental procedures were described in refs: 1, 24-28, 45, 46.

Cells were seeded at a density of 100 000 cells/cm² on a tissue culture treated surface additionally coated with Matrigel. Approximately 12 hours post-seeding; the drugs were added to the media [Lithium Chloride (Sigma) at a working concentration of 1 mM or Sodium Valproate (Sigma) at a working concentration of 0.7 mM] or kept untreated. Cellular assays and RNA extraction were performed on day 7. Experiments were performed in biological triplicates.