Camptothecin

Cells were seeded at 2000 cells per well on a 96-well plate and were grown overnight at 37 °C. The following day, cells were treated with the following drugs at indicated concentrations for 72 h for IC₅₀ assays: erastin (Cayman Chemicals), RSL3 (Apex Bio), TBH(Sigma), nutlin-3a (Calbiochem), cisplatin (Acros Organics), Etoposide (Sigma), Camptothecin (Cayman Chemicals), and doxorubicin (Cell Signaling). Because fetal bovine serum and culture media can contain significant levels of glutathione, pyruvate, and other agents that can influence sensitivity to ferroptosis (10 (link)), all ferroptosis sensitivity assays were performed in low serum/low glucose (2% fetal bovine serum, DMEM low glucose (Corning Cellgro, 1 g/l glucose)). For rescue assays, cells were pretreated with ferrostatin (Cayman Chemicals) or liproxstatin (Sigma) for 30 min prior to treatment with RSL3 for 24 h. For lipid droplet assays, cells were pretreated overnight with oleic acid (Sigma, O3008), the DGAT2 inhibitor PF-06424439 (Sigma, PZ0233), or vehicle (fatty-acid–free BSA), and viability was assayed using Trypan Blue (Corning, 25-900-CI) after 72 h. At assay endpoint, cells were treated with Alamar blue (Life Technologies Dal1025) for 3 h at 37 °C and absorbance was read using a SynergyHT plate reader (BioTek). GraphPad Prism software was used to perform the data analysis.

When organoids were passaged, 300 cells were seeded in 20 µl of Matrigel™ in a 48-well plate. Organoids were cultured as described above. Assays were done in quadruplicate for each condition and the number of organoids formed was counted 7 days post seeding. For the radio-resistance assay, organoids were irradiated at day 1 post seeding with 5 or 10 Gy and non-irradiated organoids were used as controls. For the chemo-resistance assay, organoids were incubated at day 1 post seeding with chemotherapeutic agents (30 μM cisplatin, 13119, Cayman Chemicals; 50 nM camptothecin, 11694, Cayman Chemicals; 10 μM 5-FU, 14416, Cayman Chemicals) or vehicle for 36 h.

A stock solution of the different drugs was prepared at a final concentration of 10 mM in DMSO (Sigma Cat# D8418) and stored in aliquots at −20 °C. Aurora-A kinase inhibitor (MLN8237 Cat# 13602), Polo-like kinase 4 inhibitor (CFI-400945 Cat# 16850) and Cyclin-dependent kinases inhibitor (Flavopiridol Cat# 26024) were purchased from Cayman Chemical (Ann Arbor, MI, USA). CDK4 and CDK6 inhibitor (Palbociclib Cat# HY-50767) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Triapin (Cat# S7470), Olaparib (Cat# S1060), Camptothecin (Cat# S1288) were purchased from Sellectchem (Houston, TX, USA). Each assay was performed in triplicate. Analysis of drug synergism was performed in U251 cells. Single and combined treatment of Luteolin (Sigma Aldrich Cat# L9283) and Camptothecin (Cayman Chemical Cat# S1288) or Polo-like kinase 4 inhibitor (Cayman Chemical CFI-400945 Cat# 16850) were carried out. U251 cells were plated at 2000 cells/well in a 96-well plate. 24 h later, drugs were added and cell proliferation was recorded using Incucyte^® system. Each assay was performed in triplicate.

22Rv1 cells were transfected as described above with 30 nM miR-642a-5p or miR-NC for 72 h, or treated with 10 µM Camptothecin (Cayman Chemical) for 24 h (positive control for apoptosis). Apoptosis was measured using the Annexin V-FITC Apoptois Detection Kit I (BD Biosciences, NSW, Australia), using the manufacturer’s instructions. No stain, single stain and Camptothecin treated cells were used to set gating strategies to identify live, apoptotic and dead cell populations. Samples were analysed using the BD Accuri C6 Flow Cytometer and software.