Mitotane

Organoids were grown in Matrigel™ domes within 96-well round-bottom culture plates. Recombinant human SHH was removed from the pituitary organoid growth media, 24 h prior to drug treatment. Organoids were treated with either vehicle (DMSO), cabergoline (Selleckchem S5842), ketoconazole (Selleckchem S1353), roscovitine (Selleckchem S1153), GANT61 (Stemcell Technologies 73692), pasireotide (TargetMol TP2207), mifeprostone (Selleckchem S2606), etomidate (Selleckchem S1329), mitotane (Selleckchem S1732), metyropane (Selleckchem S5416), or osilodrostat (Selleckchem S7456) at concentrations of 0, 1, 10, 100, 1000, and 10,000 nM, for 72 h. The percentage of cell viability was measured using an MTS assay (Promega G3580). Absorbance was measured at 490 nm and normalized to the vehicle. Concentrations were plotted in a logarithmic scale, and a nonlinear dose response curve regression was calculated using GraphPad Prism. An IC50 value for each drug treatment was determined based on the dose response curve, using GraphPad Prism analysis software.

For cell proliferation assay, 500 cells of 786-O or 769-P were seeded into 96-well plates containing 200 μL of medium per well. After 24 h of culture at 5% CO₂ at 37 °C, cells were exposed to 50 nM homoharringtonine (#HY-14944; MedChemExpress) for 2 days or 30 μM mitotane (#S1732; Selleckchem) for 3 days. Every 24 h, cell proliferation was measured using the CellTiter-Glo® Luminescent Cell Viability Assay kit (#G7572; Promega). For detecting the mRNA level of the targeted gene induced by drug treatment, 1 × 10⁵ 786-O or 769-P cells were seeded into each 60 mm cell culture dish. After 24 h of culture at 5% CO₂ at 37 °C, cells were treated with 50 nM homoharringtonine (HHT) for 2 days or treated with 30 μM mitotane for 3 days. Then cells were harvested for detecting the mRNA level of the targeted genes.