D2438

Explants were placed into 1.8-mL cryotubes containing 1 mL of freezing medium [10% DMSO (D2438, Sigma-Aldrich, Irvine, United Kingdom)] in foetal bovine serum, frozen at −80 °C in a CoolCell alcohol-free freezer container (Thermo Scientific, Waltham, USA) for 48 h and then kept at −80 °C in conventional storage boxes until processing (< 1 month). For thawing, cryotubes were individually incubated at 37 °C in a water bath and explants were gently washed with 1 mL of pre-warmed complete medium before been completely defrosted. Samples were twice washed with medium for DMSO removal and processed as described for fresh tissue.

First, NB SH-SY5Y cells were seeded in culture media at appropriate densities 24 h before treatment. Next, the cells were treated with physiological (100 pM, 1 nM, and 10 nM) and pharmacological (100 nM, 1 μM, and 10 μM) concentrations of 2-ME2. The investigations were carried out in a medium without FBS in order to completely exclude the influence of hormones derived from sera. The solvent used to prepare 2-ME2 solutions, DMSO (dimethyl sulfoxide, D2438, Sigma Aldrich, Poland), was provided to control cells in the same ratio. The final DMSO concentration in the incubation medium was less than 0.1%.

All compounds were purchased from commercial vendors. All compounds were made at 1000 × stocks in DMSO (Sigma Aldrich #D2438), except for sodium valproate, which was dissolved in ddH2O. NPCs and neurons were generated and maintained as described above. For treatment, the stock compounds were diluted 1:1000 in NPC media (final DMSO concentration 0.1%), which was then applied to confluent NPCs or 18-day differentiated neurons derived from confluent NPC cultures for indicated time periods.

Three HPV-positive (UPCI-SCC-154, UM-SCC-104 and UM-SCC-47) and three HPV-negative (SQD9, SCC61 and CAL27) HNSCC cell lines were used as described previously [6 (link)]. All cell lines were authenticated by ATCC via short-tandem repeat profiling, and all experiments were performed with mycoplasma-free cells. AZD6738 was bought from Selleck Chemicals (S7693, Houston, TX, USA), and for in vitro testing, a stock solution of 10 mM was made in dimethylsulfoxide (DMSO) (D2438, Sigma-Aldrich, Saint Louis, MO, USA). For in vivo use, AZD6738 was dissolved in 10% DMSO, 40% propylene glycol (202398, Sigma-Aldrich, Saint Louis, MO, USA), and 50% sterile dH₂O.

4-OHT (Sigma; catalog #H6278) was freshly dissolved in warmed ethanol at a concentration of 20 mg/ml on each experimental day. Once dissolved, a volume of corn oil (Acros Organics; catalog #8001-30-7) equivalent to the volume of ethanol was added. The mixture was heated and spun at 1725 rpm in a Speedvac concentrator (Savant; model #SVC-100H) for 20 min. The 4-OHT/corn oil solution was then loaded into insulin syringes (BD; catalog #329461) at a dose of 50 mg/kg for injection. Clozapine-n-oxide (CNO; Tocris; catalog #4936) was dissolved in 0.5% DMSO (Sigma; catalog #D2438; v/v in 0.9% sterile saline) at room temperature to a concentration of 3.79 mg/ml. The solution was then aliquoted and stored at −20°C until experimentation. A dose of 2.5 mg/kg was used for injection; 0.5% DMSO (v/v in 0.9% sterile saline) was used as a vehicle control injection.

Cell monolayers were cultured for three additional days without TSH (5H medium) before stimulation and addition of DE-71 in serum free medium for 72 hours. DE-71 (pentaBDE, lot 7550K20A was kindly provided by Martha Axelstad, National Food Institute, Technical University of Denmark and Dr. Kevin Crofton of the U.S. Environmental Protection Agency), was dissolved in dimethyl sulfoxide (DMSO, D2438 Sigma-Aldrich, St. Louis, MO, USA) prior to dissolving in culture medium without FBS, resulting in final concentrations of 0.01, 0.1, 1, 5, 10 and 50 mg DE-71/L, respectively, and 1 ‰ DMSO in the cell cultures. Three negative controls (without DE-71) were included on each culture plate: one non-stimulated (5H medium) without DMSO and two TSH-stimulated (6H medium) cell populations, respectively, with and without 1 ‰ DMSO added. All experiments were conducted in duplicate in the culture plates. The cell function is dependent on TSH, which was assessed by comparing 6H and 5H controls. Cells were visually inspected by light microscopy at the end of incubation. Cell supernatants were harvested and stored at -20°C until analyses. The cells were harvested on ice, immediately after removal of the cell supernatant using lysis buffer (Qiagen, Hilden, Germany) and stored at -80°C with prior addition of 70% ethanol (350 μL pr. well).