Ketoconazole

The HCC cell lines were purchased from Shanghai Cell Bank. Acquired radio-resistant cell lines MHCC97H IR-R and MHCC97L IR-R were constructed as follows [13 (link)]. These cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gbico, America) and maintained at 37 °C and 5% CO₂. Lentiviral vectors and short hairpin RNA were designed and constructed by WZ Biotech (Jinan, China). Flag-tagged HK2 overexpression vectors (LV-HK2) and control vectors (LV-RFP) were transferred into MHCC97H, MHCC97L, and H22. Consistently, HK2 shRNA (shHK2) and control short hairpin RNA (shGFP) were transfected into MHCC97H IR-R, MHCC97L IR-R, and QGY7701. AIMP2 overexpression vectors (LV-AIMP2) and control vectors (LV-NC) were transferred into MHCC97H (LV-RFP&LV-HK2) and MHCC97L (LV-RFP&LV-HK2) cells. The small interfering RNA (siRNA) oligonucleotides constructed by Ribobio (Guangzhou, China) contain siAIMP2 (three targets) and siControl (siNC). The plasmid of mCherry-GFP-LC3 II was constructed by Hanyi Biotech (Guangzhou, China). Both were transfected into cells using Lipofectamine 3000 (Thermo Fischer Scientific) transfection reagent following the supplier’s recommendations. Ketoconazole, chloroquine (CQ) and MG-132 were all purchased from Selleck and dissolved in DMSO.

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.

Neratinib (catalogue no. S2150), nilotinib (catalogue no. S1033), ketoconazole (catalogue no. S1353), amiodarone HCl (catalogue no. S1979) and fluticasone propionate (catalogue no. S1992) were obtained from Selleckchem, Methadone stock solution (1 mg/ml) was provided by Department of Chemistry, University of Oslo. Stock solutions of drugs were prepared in DMSO in concentration 10 mM. 3D PHH (at day 7 after thawing) and 3D iHLC (after 24 days of differentiation) were incubated with indicated compounds for 24 h and 48 h, diluted in the concentration 10 μM in serum-free William's E media, supplemented with 1 % (v/v) ITS, 0.1 μM Dexamethasone. Control organoids were incubated in the same medium with 0.1 % (v/v) DMSO.