Abstract
Ferroptosis is a form of cell death involving the formation of lipid peroxyl radicals, with potential therapeutic applications. Sensitivity to ferroptosis is expected to vary in different organelles. To monitor in real time the onset and progression of lipid peroxidation in ferroptosis, here we report lipophilic fluorogenic radical-trapping antioxidants, embedding in endoplasmic reticulum, lysosomes, mitochondria and plasma membrane. We show that endoplasmic reticulum- and lysosome-embedding fluorogenic radical-trapping antioxidants are most effective in protecting from cell death. The onset of lipid peroxidation happens in the endoplasmic reticulum, with lipid hydroperoxide accumulating in Golgi-associated vesicles. Disintegration of these structures spreads lipid hydroperoxide intracellularly, acting as ‘free radical embers’. Outwards migration of oxidized lipids to plasma membrane, the ultimate sink for oxidized lipids, was recorded. Our results underscore Golgi-associated structures as a site to regulate ferroptosis progression. The work further positions fluorogenic radical-trapping antioxidants as valuable tools for unravelling the dynamic subcellular progression of ferroptosis.
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All data are available within the article and the Supplementary Information. Source data are provided with this paper.
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The macro plugin for calculating the CTCF is provided as Supplementary Software.
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Acknowledgements
G.C. is grateful to the National Science and Engineering Research Council of Canada (NSERC) and the Canadian Foundation for Innovation for funding. L.X. is grateful to FRQNT, NSERC CREATE PROMOTE and CRBS for postgraduate scholarships. W.Z. and J.M. are grateful to NSERC and FRQNT for postgraduate scholarships. T.C.L. is grateful to NSERC for post-doctoral scholarships. J.F.S.T. and D.H. are grateful to FRQNT for postgraduate scholarships. The funders had no role in study design, data collection and analysis, the decision to publish or the preparation of the manuscript. We thank A. Van Kessel for his insightful discussion, J. Ramos-Sanchez for his help on fluorescence lifetime measurements and N. Hardt for her help with fluorescence imaging experiments. We thank Columbia University, Helmholtz Zentrum München and University of Ottawa for providing the cell lines used in this study.
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L.X. and W.Z. contributed equally. G.C. conceived the study. L.X., W.Z. and G.C. wrote the manuscript. L.X., J.M. and T.C.L. designed and performed the synthesis. J.F.S.T. and W.Z. performed cell viability assay. L.X. and W.Z. performed fluorescence imaging experiments and data analysis. J.F.S.T. and D.H. performed data analysis. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
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Extended data
Extended Data Fig. 1 Photophysical properties of fluorogenic RTAs and their corresponding fluorescent control analogues in acetonitrile.
a. Extinction coefficient, fluorescence quantum yield, and fluorescence lifetimes. The fluorescence lifetimes for lyso-H4B-PMHC and PM-H4B-PMHC have two components, corresponding to the reduced form (short lifetime, major component) and trace impurities of the oxidized form (long lifetime, minor component), respectively. Fluorescence lifetimes of mito-H5B-PMHC and mito-H5B-CH3 were obtained using 496 nm excitation and emission at 520 nm were monitored. Fluorescence lifetimes of lyso-H4B-PMHC, lyso-H4B-CH3, PM-H4B-PMHC, and PM-H4B-CH3 were obtained using 515 nm excitation and emission at 525 nm were monitored. For mito-H5B-PMHC, only a long lifetime corresponding to the oxidized form was detected, whereas the quantum yield is 1/60 of that of the fluorescent analogue mito-H5B-CH3. We postulate that lifetime of the reduced form is too short to be measured, as PeT from PMHC to a mono-substituted BODIPY moiety is more efficient than to a di-substituted BODIPY moiety. b. Emission profile of the fluorogenic RTAs in acetonitrile and upon addition of MeO-AMVN (V70), where peroxyl radicals were generated via thermolysis. In these experiments, the emission profile of the fluorogenic RTAs (100 μM) was first monitored in acetonitrile for 1 minute (grey curve). V70 (20 mM) was then added, and the emission profile was monitored for the subsequent 59 minutes.
Extended Data Fig. 2 Validating targeting specificity and cellular uptake with fluorescent control compounds.
The targeting specificity of fluorogenic RTAs cannot directly validated by colocalization studies, as they can only be visualized after reacting with lipid peroxyl radicals. a-e. 100x HILO images of HT-1080 cells co-stained with H4B-CH3 (10 nM) and either ERTracker Red (1 μM, a) or LipidSpot 610 (1x, b) to validate ER and lipid droplets accumulation, respectively, of H4B-PMHC. c) mito-H5B-CH3 (10 nM) and mitoTracker DR (25 nM) to show mitochondrial localization of mito-H5B-PMHC. d) lyso-H4B-CH3 (10 nM) and lysoTracker DR (75 nM) to show lysosomal localization of lyso-H4B-PMHC. e) PM-H4B-CH3 (100 nM) in HT-1080 cells transfected with PM-RFP to show PM localization of PM-H4B-PMHC. The region within the yellow outline was used to calculate Mander’s and Pearson’s coefficients. In each experiment, cells stained with the commercial organelle marker were incubated with the fluorescent control compound for 10 minutes, before colocalization images were captured. Each experiment was conducted once. f) The intensity of the 488-channel along the indicated line is plotted against that of the 561 channel to show colocalization of PM-H4B-CH3 with PM-RFP. g) The cell outline (blue) was identified via DIC channel and was plotted against the intensity of the 488-channel along the indicated line. Scale bar = 12 µm. h) Mander’s coefficients (M1 and M2) and Pearson’s coefficient of the fluorescent control compounds with commercial organelle markers. i) Time course of partition profile of the fluorescent control compounds in HT-1080 cells. Imaging was initiated immediately after addition of the fluorescent control compound. Bars = SEM, n = 4 (replicates across independent wells).
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.
Extended Data Fig. 4 Temporal correlation of the onset and progression of lipid peroxyl radical production, cell morphology change and membrane permeability at basal conditions, with RSL3 (1 or 0.1 µM), or with Erastin (10 µM) (replicates of Fig. 2b).
a-n. Lipid peroxyl radical generation was recorded by fluorogenic RTA fluorescence (10 nM, green curve). Cell rounding was marked visually by time of blebbing (black curve). Membrane integrity assessed by PI (1 µM, orange curve) fluorescence. Scale bar = 64 µm. Each condition was twice on different days with similar results. o-p. Comparison of the profiles of HT-1080 morphology change in the presence of RSL3 and different RTAs.
Extended Data Fig. 5 Parameters describing CTCF time trajectories for fluorogenic RTAs and the time evolution of cell morphology change.
The data was recorded in the presence of RSL3 (1 μM or 0.1 μM) or Erastin (10 μM). Values are expressed as mean ± s.d., n = 4 (replicates across independent wells).
Extended Data Fig. 6 Evaluation of ferroptosis inhibition potency for competing RTAs (resveratrol, left; PMHC, center; phenoxazine, right).
Each of these antioxidants may be ranked based on their autooxidation inhibitory effect as, respectively: weaker than, as strong as, and stronger than H4B-PMHC. Lipid peroxyl radical measured by H4B-PMHC fluorescence (10 nM, top). Membrane integrity assessed by PI fluorescence (1 µM, bottom). For each condition, the competing RTA was added together with H4B-PMHC and incubated for 10 minutes. Imaging was initiated immediately after RSL3 addition (1 μM). For control groups (basal conditions, grey), only H4B-PMHC were added (no competing RTA or RSL3) but conducted under otherwise similar conditions. Bars = SEM, n = 4 (replicates across independent wells). Imaging studies with phenoxazine, an RTA with superior scavenging activity than α-tocopherol (and thus H4B-PMHC), showed suppression of fluorescence enhancement. In turn, imaging with PMHC, which has a comparable RTA activity to H4B-PMHC, displayed a flattening of the runaway period in a concentration dependent manner. Imaging with resveratrol, a case study for a poor RTA, lead to no changes in the induction period, indicating that H4B-PMHC outcompeted resveratrol as an RTA.
Extended Data Fig. 7 Identification of punctuate structures observed in ferroptotic HT-1080 cells stained with H4B-PMHC.
a-c. Colocalization timelapse (100x HILO) in HT-1080 cells stained with H4B-PMHC (10 nM, cyan), co-stained with LipidSpot 610 (a, magenta), lysoTracker DR (b, magenta), or mitoTracker DR (c, magenta). Imaging was conducted upon RSL3 (1 µM) addition. Scale bars = 12 µm. Scale bar of the enlarged region = 4 µm. Colocalization of all channels is shown in the “merged” panel. Each experiment was conducted once. In each experiment, 4 FOVs were monitored. d. To quantify the contribution to CTCF from these punctuate structures in Fig. 3b, the outline of these structures and that of the whole cell were identified via CellProfiler. Next, the integrated intensity and total area for punctate structures and the whole cell were determined, and intensity-time trajectories of these two regions were plotted (also see Fig. 3e). e. Regions corresponding to Golgi-associated vesicles (cyan and yellow) and lipid droplets or Golgi (magenta), were identified by CellProfiler. The vesicular structures do not colocalize with lipid droplets but were formed next to the Golgi apparatus. Scale bars = 12 µm.
Extended Data Fig. 8 High magnification imaging of HT-1080 cells with H4B-PMHC under a range of conditions.
a. Colocalization (100x HILO) of H4B-PMHC (10 nM, cyan) with ER Tracker Red (1 µM, magenta). Imaging was conducted upon Cu (II) and cumene hydroperoxide addition. Experiment was conducted once, with 4 FOVs monitored. b. Colocalization (100x HILO) of H4B-PMHC (10 nM, cyan) with ER Tracker Red (1 μM, not shown due to photobleaching of the dye during long time-lapse of imaging). Imaging was conducted upon RSL3 (0.1 μM) addition. Scale bars = 12 μm. Scale bar of the enlarged region = 4 μm. Colocalization of all channels (H4B-PMHC, organelle-tracker (only for a), and DIC) is shown in the “merged” panel. Experiment was repeated twice on different days with similar results. In experiment, 4 FOVs were monitored.
Extended Data Fig. 9 High magnification imaging of HT-1080 cells either with fluorescent control compounds in the presence of RSL3 or with Erastin and mito-H5B-PMHC.
a. Colocalization (100x HILO) of H4B-CH3 (10 nM, cyan) with LipidSpot 610 (1x, magenta). b. Colocalization (100x HILO) of mito-H5B-CH3 (10 nM, cyan) with mitoTracker DR (25 nM, magenta). c. Colocalization (100x HILO) of lyso-H4B-CH3 (10 nM, cyan) with lysoTracker DR (75 nM, magenta). Imaging was conducted upon RSL3 addition (1 μM). d. Colocalization (100x HILO) of mito-H5B-PMHC (10 nM, cyan) with mitoTracker DR (25 nM, magenta). Imaging was conducted upon Erastin addition (10 μM). Scale bars = 12 μm. Colocalization of all channels are shown in the “merged” panel. Each experiment was conducted once, with 4 FOVs monitored.
Extended Data Fig. 10 Observations in Pfa-1 cells are consistent with those in HT-1080 cells.
a-c. Temporal correlation of the onset and progression of lipid peroxyl radical production, cell morphology change and membrane permeability at basal conditions, with RSL3 (1 µM), or 48 hours after TAM (1 µM) treatment (replicates of Fig. 2h-i). Lipid peroxyl radical generation was recorded via H4B-PMHC enhancement (10 nM, green curve). Cell death was marked visually by the time of blebbing (black curve). Membrane integrity was assessed by PI (1 µM, orange curve) fluorescence. Each experiment was repeated twice on different days with similar results. d. Tabulated values of parameters describing CTCF of fluorogenic RTAs and morphology change curves in the presence of RSL3 (1 μM) or 48 hours after TAM (1 μM) treatment. e-f. Colocalization (100x HILO) of H4B-PMHC (10 nM, cyan) with ER Tracker Red (1 μM, not shown due to poor staining) in GPX4 proficient Pfa-1 cells with RSL3 (1 μM) treatment (e) or GPX4 KO Pfa-1 cells (f). Scale bars = 12 μm. Scale bar of the enlarged region = 4 μm. Colocalization of all channels (H4B-PMHC and DIC) is shown in the “merged” panel. Each experiment was repeated twice on different days with similar results. In each experiment, 4 FOVs were monitored.
Supplementary information
Supplementary Information
Supplementary methods for synthesis, NMR and HRMS spectra and Discussion
Supplementary Video 1
Temporal correlation of the onset and progression of lipid peroxyl radical production, cell morphology change, and membrane permeability in HT-1080 cells, imaged under 20x widefield configuration. Fluorescence enhancement of H4B-PMHC (10 nM), fluorescence enhancement of PI (1 μM), and cell morphology are monitored via RTA channel, PI channel, and DIC, respectively. Scale bar = 64 μm. Ferroptosis was initiated at t = 0 min by addition of RSL3 (1 μM). Representative timelapses are shown in Fig. 2a.
Supplementary Video 2
Colocalization of H4B-PMHC (10 nM, cyan) with ER, lipid droplets, and Golgi markers (magenta) in HT-1080 cells, imaged under 100x HILO modality. Ferroptosis was initiated at t = 0 min by addition of RSL3 (1 μM). Scale bar = 12 μm. Representative timelapses are shown in Fig. 3b-c and Extended Data Fig. 7a.
Supplementary Video 3
Colocalization of mito-H5B-PMHC (10 nM, top row, cyan) and mito-H5B-CH3 (10 nM, bottom row, cyan) with mitoTracker Deep red (25 nM, magenta) in HT-1080 cells, imaged under HILO modality. Ferroptosis was initiated at t = 0 min by addition of RSL3 (1 μM). Scale bar = 12 μm. Representative timelapses are shown in Fig. 4a and Extended Data Fig. 9b.
Supplementary Video 4
Colocalization of lyso-H4B-PMHC (10 nM, top row, cyan) and lyso-H4B-CH3 (10 nM, bottom row, cyan) with lysoTracker Deep red (75 nM, magenta) in HT-1080 cells, imaged under HILO modality. Ferroptosis was initiated at t = 0 min by addition of RSL3 (1 μM). Scale bar = 12 μm. Representative timelapses are shown in Fig. 4b and Extended Data Fig. 9c.
Supplementary Video 5
Colocalization of PM-H4B-PMHC (100 nM, cyan) and PM-RFP (magenta) in HT-1080 cells, imaged under HILO modality. Ferroptosis was initiated at t = 0 min by addition of RSL3 (1 μM). Scale bar = 12 μm. Representative timelapses are shown in Fig. 4c.
Supplementary Software 1
Code for ImageJ used to quantify average fluorescence intensity per cell in 20× movies.
Source data
Source Data Fig. 1
Raw data for cell viability assays in presence of RSL3 and Erastin (Fig. 1g,h).
Source Data Fig. 3
Raw data for quantifying fluorescence enhancement of H4B-PMHC in Fig. 3e,f.
Source Data Fig. 4
Raw data for quantifying fluorescence enhancement of mito-, lyso- and PM-fluorogenic RTAs (Fig. 4d).
Source Data Extended Data Fig./Table 1
Raw data for fluorescence enhancement of mito-, lyso- and PM-fluorogenic RTAs in presence of V70 (Extended Data Fig. 1b).
Source Data Extended Data Fig./Table 4
Raw data for fluorescence enhancement and cell morphology change in presence of RSL3 or Erastin (replicates of Fig. 2b).
Source Data Extended Data Fig./Table 10
Raw data for fluorescence enhancement and cell morphology change of Pfa-1 cells, either GPX4 proficient with RSL3 treatment or GPX4 depleted (replicates of Fig. 2h,i).
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Xu, L., Zhang, W., Sánchez Tejeda, J.F. et al. Live-cell imaging with fluorogenic radical-trapping antioxidant probes reveals the onset and progression of ferroptosis. Nat. Chem. (2025). https://doi.org/10.1038/s41557-025-01966-x
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DOI: https://doi.org/10.1038/s41557-025-01966-x
