Extended Data Fig. 3: Fluorogenic RTAs show different efficacy in ferroptosis inhibition.
a-d. HT-1080 cell viability assessed via resazurin (normalized resazurin intensity), varying incubation time and/or RTA dose (as compared to Fig. 1g). Cells were treated with 0–1000 nM RSL3 and a) 10 nM fluorogenic RTAs for 3 hours, b) 10 nM fluorogenic RTAs for 5 hours, c) 10 nM fluorogenic RTAs for 24 hours, or d) 100 nM fluorogenic RTAs for 3 hours. In control experiments, no RTAs were added (white) but conducted under otherwise the same conditions. For each condition, bars = s.d., n = 3 (replicates across independent wells). e. Impact of fluorogenic RTAs on RSL3 EC50 values (expressed as mean ± s.d.), extracted from a-d. We note that although the protection hierarchy is not dependent on the RTA dose, difference in the efficiency of ferroptosis inhibition was most pronounced in presence of higher RTA dose (100 nM). In turn, the effective EC50 of RSL3 decreased with increasing incubation time. f-i. Dependence of rate of lipid peroxyl radical generation on RTA dosing. Rate of lipid peroxidation measured by 10 and 100 nM of H4B-PMHC (f), mito-H5B-PMHC (g), lyso-H4B-PMHC (h), or PM-H4B-PMHC (i) fluorescence in presence of 1 µM RSL3. Scale bar = 64 µm. While in the presence of 10 nM H4B-PMHC, mito-H5B-PMHC, or lyso-H4B-PMHC, after RSL3 addition, HT-1080 cells exhibited a short induction period prior to fluorescence enhancement and characteristic ferroptosis morphology change, with 100 nM of these fluorogenic RTAs, ferroptosis was inhibited over 3 h. In contrast, cells treated with both 10 and 100 nM PM-H4B-PMHC showed the same induction period and underwent ferroptosis within the 3-hour time window.
