APD-CLDs induce a hypoxia- and ETC/OXPHOS-dependent necrotic cell death.
Description
Realtime apoptosis-necrosis assay: PANC-1 cells were treated with BE-43547A2 (3 µM) or the apoptosis-inducer raptinal (10 µM) under either hypoxia or normoxia while the membrane integrity and phosphatidylserine translocation was followed in real-time using a membrane impermeable DNA stain and an annexin V-luciferase fusion protein, respectively (RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay, Promega, Cat#: JA1011). A delay between phosphatidylserine translocation and loss of membrane integrity designates a canonical apoptotic cell death. Data is represented in relative fluorescent and luminescent units. N = 3. Inhibition of cell death pathways: BE-43547A2-induced cell death in hypoxic PANC-1 cells cannot be blocked by the pan-caspase inhibitor Q-VD-OPh (24 h). As a positive control normoxic cells treated with the apoptosis-inducer raptinal could be rescued with Q-VD-OPh. Toxicity was measured by CellTox Green (Promega) and RFUs were normalized to the average RFU of untreated cells. N = 3. PANC-1 cells were treated with BE-43547A2 (100 nM) in combination with serial dilutions of necrostatin-5 (Nec-5) or DPQ for 48 hours under hypoxia. Toxicity was measured by CellTox Green. N = 3. Modulatory profiling: PANC-1 cells were treated with lethal concentrations of APD-CLDs or ferroptosis-inducers in combination with a panel of small molecule modulators of various cellular processes. Each data point represents the mean of three replicates. Viability was assessed with CellTiter Blue (Promega, Cat# G8080). Hits in the modulatory profiling were confirmed by treating PANC-1 cells with serial dilutions of APD-CLDs or ferroptosis-inducers in the absence or presence of the hit compound for 48 hours under hypoxia. N = 3. PANC-1 cells treated with APD-CLDs can be rescued by the ER stress inhibitor azoramide. N = 3. Full dose-response curves of the modulation of ferroptosis with the ferroptosis-inhibitors NHI-2 and antimycin A. N = 3.