Journal: Nature Cell Biology

Article Title: Endoplasmic reticulum–mitochondria contacts are prime hotspots of phospholipid peroxidation driving ferroptosis

doi: 10.1038/s41556-025-01668-z

Figure Lengend Snippet: a , A schematic representation of 9xL spacer. b , Representative volume-rendered 3D reconstructions of confocal z -stacks from time-lapse live images of MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer and the ER marker BFP–KDEL (cyan) and stained with MitoTracker Far Red (magenta) and Liperfluo (green) at time 0, 5, 15 and 30 min after RSL3 (0.5 µM). The EMCS masked volume, representing colocalization of the ER and mitochondria, is indicated in white. The magnified images represent, respectively, (i) the merge of ER, mitochondrial surfaces and EMCS mask and (ii) Liperfluo and mitochondrial surfaces together with EMCS mask. Scale bar, 10 µm. Zoom, 1 µm. c , A quantification of the per cent of EMCSs normalized on the ER volume (BFP–KDEL) at time 0, 5, 15 and 30 min after RSL3 (0.5 µM) in MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer ( n = 3 biological replicates). ctrl, control. d – f , A colocalization (Mander’s coefficient (M1), per cent) of Liperfluo in the EMCS ( d ), mitochondria ( e ) and ER ( f ) masks at time 0, 5, 15 and 30 min after RSL3 (0.5 µM) treatment in MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer ( n = 3 biological replicates, the same cells ( n = 7 mRFP control, n = 7 9xL spacer) were acquired and analysed at each timepoint per condition). Similar experiments and settings have been reproduced independently on Fig. , Supplementary Fig. and Supplementary Fig. . All quantitative data are the mean ± s.e.m. In c , d , e and f , the statistical significance was determined by a two-way repeated-measures ANOVA, Tukey post hoc test. n.s., not significant ( P > 0.05), * P ≤ 0.05, ** P < 0.01, *** P < 0.001. Panel a created with BioRender.com .

Article Snippet: The reagents used were: Bodipy 581/591 C11 (Thermo Fisher Scientific); Liperfluo (Dojindo), d -glucose (Sigma-Aldrich, G7021-1KG); DAPI (Thermo Fisher Scientific, 62248); DAPI (Thermo Fisher Scientific, D1306); DMEM—high glucose (Sigma-Aldrich, D0422); Gibco DMEM/F-12 (Thermo Fisher Scientific, 11320074); Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich, DUO82040); GSK2606414 PERK Inhibitor (Toronto Research Company, G797800); MitoPerox (Abcam, ab146820); MitoTracker Far Red (Thermo Fisher Scientific, M22426 ); MitoTracker Green (Thermo Fisher Scientific, M7514); SE Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XC-1024); P3 Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XP-3024); penicillin and streptomycin (Sigma-Aldrich, P0781); Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106×4); Pierce Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific, 88266); potassium chloride (Janssen Chimica, 7447407); protease inhibitor (Thermo Fisher Scientific, A32953); saponin (Sigma-Aldrich, 8047152); Gibco, Roswell Park Memorial Institute Sodium Chloride, (Sigma-Aldrich, A0431796); SYTOX Green (Sigma-Aldrich, S7020); TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR 6000); Tris base (Sigma-Aldrich, 77861); Triton (Sigma-Aldrich, T9234); Tween (Sigma-Aldrich, P4780).

Techniques: Transfection, Control, Marker, Staining

Journal: Nature Cell Biology

Article Title: Endoplasmic reticulum–mitochondria contacts are prime hotspots of phospholipid peroxidation driving ferroptosis

doi: 10.1038/s41556-025-01668-z

Figure Lengend Snippet: a , Representative 2D images of MEFs transiently transfected with the ER marker BFP-KDEL (cyan) and stained with MitoTracker Far Red (magenta) and Bodipy C11 (green) at time 0 and at 5’, 15’, 30’ after RSL3 (0.5 µM). EMCS masked area, representing colocalization of the ER and mitochondria, is indicated as white. Magnified images represent respectively ox-Bodipy together with i) ER surface, ii) EMCS mask and iii) mitochondrial surfaces together with EMCS mask. Scale bar, 10 µm. Zoom, 1 µm. b-e , Colocalization (% M1, Mander’s coefficient) of oxidized Bodipy C11 (ox-Bodipy C11) (green) signal in the ER, EMCS mask, mitochondria and the residual cellular region outside the ER + mitochondrial masks, at time 0 and 5, 15, 30‘ after RSL3 (0.5 µM). (n = 3 biological replicates). f , Total Cell Liperfluo intensity normalized on total cellular volume (MFI) at time 0 and 5, 15, 30‘ after RSL3 (0.5 µM). (n = 3 biological replicates). g , Total Cell ox-Bodipy C11 intensity (green) normalized on non-oxidized Bodipy C11 (red) at time 0 and 5, 15, 30‘ after RSL3 (0.5 µM). (n = 3 biological replicates). All quantitative data are mean ± SEM. In b-e, g , statistical significance was determined by one-way ANOVA, Dunnett post-hoc test. In f , statistical significance was determined by RM one-way ANOVA, Dunnett post-hoc test. NS, not significant (P > 0.05), *P ≤ 0.05, **P < 0.01, ***P < 0.001. Source numerical and statistical data are provided.

Article Snippet: The reagents used were: Bodipy 581/591 C11 (Thermo Fisher Scientific); Liperfluo (Dojindo), d -glucose (Sigma-Aldrich, G7021-1KG); DAPI (Thermo Fisher Scientific, 62248); DAPI (Thermo Fisher Scientific, D1306); DMEM—high glucose (Sigma-Aldrich, D0422); Gibco DMEM/F-12 (Thermo Fisher Scientific, 11320074); Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich, DUO82040); GSK2606414 PERK Inhibitor (Toronto Research Company, G797800); MitoPerox (Abcam, ab146820); MitoTracker Far Red (Thermo Fisher Scientific, M22426 ); MitoTracker Green (Thermo Fisher Scientific, M7514); SE Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XC-1024); P3 Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XP-3024); penicillin and streptomycin (Sigma-Aldrich, P0781); Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106×4); Pierce Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific, 88266); potassium chloride (Janssen Chimica, 7447407); protease inhibitor (Thermo Fisher Scientific, A32953); saponin (Sigma-Aldrich, 8047152); Gibco, Roswell Park Memorial Institute Sodium Chloride, (Sigma-Aldrich, A0431796); SYTOX Green (Sigma-Aldrich, S7020); TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR 6000); Tris base (Sigma-Aldrich, 77861); Triton (Sigma-Aldrich, T9234); Tween (Sigma-Aldrich, P4780).

Techniques: Transfection, Marker, Staining

Journal: Nature Cell Biology

Article Title: Endoplasmic reticulum–mitochondria contacts are prime hotspots of phospholipid peroxidation driving ferroptosis

doi: 10.1038/s41556-025-01668-z

Figure Lengend Snippet: a , A schematic representation of Mander’s coefficient (M1). The M1 quantifies the fraction of the signal intensity of the lipid peroxidation probe that colocalizes in each subcellular compartment/organelle relative to the total signal intensity of the probe. b , Representative volume-rendered 3D reconstructions of confocal z -stacks from time-lapse live images of MEFs transiently transfected with ER marker Sec61ß–mCherry (cyan) and stained with MitoTracker Far Red (magenta) and Liperfluo (green) at time 0 and 5, 15 and 30 min after RSL3 (0.5 µM). The EMCS masked volume, representing colocalization of ER and mitochondria, is indicated in white. The magnified images represent, respectively, (i) Liperfluo and mitochondrial surfaces, (ii) Liperfluo surface and EMCS mask and (iii) Liperfluo and mitochondrial surfaces together the with EMCS mask. Scale bar, 10 µm. Zoom, 1 µm. c – e , The colocalization (M1%) of Liperfluo in the EMCSs ( c ), mitochondria ( d ) and ER ( e ) masks at time 0 and 5, 15 and 30 min after RSL3 (0.5 µM) ( n = 3 biological replicate, the same cells ( n = 11) were acquired and analysed at each timepoint per condition). Similar experiments and settings have been reproduced independently in Fig. , Supplementary Fig. and Supplementary Fig. . f , g , The per cent of total PE ( f ) and PC ( g ) in the input, EMCSs and mitochondrial fraction ( n = 3 biological replicates, 105 PE subspecies, 58 PC subspecies). h – k , The per cent of PEox ( h ) and PCox ( j ) in the input, EMCSs and mitochondrial fraction and PEox ( i ) and PCox ( k ) fold changes compared with corresponding conditions cotreated with Fer-1 (1 µM) ( n = 3 biological replicates, 25 PEox subspecies, 29 PCox subspecies). i , A schematic representation of the complementary approaches and methodology used to assess the dynamics of early interorganelle PLox transfer. All quantitative data are the mean ± s.e.m. In c – e , the statistical significance was determined by a repeated-measures one-way ANOVA, Dunnett post hoc test. In f – h and j , the statistical significance was determined by a repeated-measures one-way ANOVA, Tukey post hoc test. In i and k , the statistical significance was determined by a one-way ANOVA, Tukey post hoc test. n.s., not significant ( P > 0.05), * P ≤ 0.05, ** P < 0.01, *** P < 0.001. Panel a created with BioRender.com .

Article Snippet: The reagents used were: Bodipy 581/591 C11 (Thermo Fisher Scientific); Liperfluo (Dojindo), d -glucose (Sigma-Aldrich, G7021-1KG); DAPI (Thermo Fisher Scientific, 62248); DAPI (Thermo Fisher Scientific, D1306); DMEM—high glucose (Sigma-Aldrich, D0422); Gibco DMEM/F-12 (Thermo Fisher Scientific, 11320074); Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich, DUO82040); GSK2606414 PERK Inhibitor (Toronto Research Company, G797800); MitoPerox (Abcam, ab146820); MitoTracker Far Red (Thermo Fisher Scientific, M22426 ); MitoTracker Green (Thermo Fisher Scientific, M7514); SE Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XC-1024); P3 Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XP-3024); penicillin and streptomycin (Sigma-Aldrich, P0781); Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106×4); Pierce Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific, 88266); potassium chloride (Janssen Chimica, 7447407); protease inhibitor (Thermo Fisher Scientific, A32953); saponin (Sigma-Aldrich, 8047152); Gibco, Roswell Park Memorial Institute Sodium Chloride, (Sigma-Aldrich, A0431796); SYTOX Green (Sigma-Aldrich, S7020); TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR 6000); Tris base (Sigma-Aldrich, 77861); Triton (Sigma-Aldrich, T9234); Tween (Sigma-Aldrich, P4780).

Techniques: Transfection, Marker, Staining

Journal: Nature Cell Biology

Article Title: Endoplasmic reticulum–mitochondria contacts are prime hotspots of phospholipid peroxidation driving ferroptosis

doi: 10.1038/s41556-025-01668-z

Figure Lengend Snippet: a , Representative volume-rendered 3D reconstructions of confocal z -stacks from time-lapse live images of MEFs transiently transfected with the ER marker Sec61ß-mCherry (cyan) and stained with MitoTracker Far Red (magenta) and Liperfluo (green) at time 0 and 5’, 15’, 30’ after IKE (0.625 µM). EMCS masked volume, representing colocalization of the ER and mitochondria, is indicated as white. Magnified images represent respectively, i) merge of ER, mitochondrial surfaces and EMCS mask and ii) Liperfluo and mitochondrial surfaces together with EMCS mask. Scale bar, 10 µm. Zoom, 1 µm. b-d , Colocalization (% M1, Mander’s coefficient) of Liperfluo in the EMCS ( b ), mitochondria ( c ), ER ( d ) masks at time 0 and 5’, 15’, and 30’ after IKE (0.625 µM) without or with Fer-1 (1 µM) in MEFs. (n = 3 biological replicates). e , Quantification of the % EMCS normalized on the ER volume (Sec61ß-mCherry) at time 0 and 5’, 15’, 30‘ after IKE (0.625 µM) and cotreatment with Fer-1 (1 µM) in MEFs. f , Total Cell Liperfluo intensity normalized on total cellular volume (MFI) at time 0 and 5, 15, 30 ‘after IKE (0.625 µM) and cotreatment with Fer-1 (1 µM) in MEFs. (n = 3 biological replicates). (n = 3 biological replicates, the same cells (n = 11 IKE, n = 9 IKE+Fer-1) were acquired and analyzed at each timepoint per condition). Similar experiments and settings have been reproduced independently in Fig. , Fig. and Supplementary Fig. . All quantitative data are mean ± SEM. In b-f , statistical significance was determined by one-way ANOVA, Sidak post-hoc test. NS, not significant (P > 0.05), *P ≤ 0.05, **P < 0.01, ***P < 0.001. Source numerical and statistical data are provided.

Article Snippet: The reagents used were: Bodipy 581/591 C11 (Thermo Fisher Scientific); Liperfluo (Dojindo), d -glucose (Sigma-Aldrich, G7021-1KG); DAPI (Thermo Fisher Scientific, 62248); DAPI (Thermo Fisher Scientific, D1306); DMEM—high glucose (Sigma-Aldrich, D0422); Gibco DMEM/F-12 (Thermo Fisher Scientific, 11320074); Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich, DUO82040); GSK2606414 PERK Inhibitor (Toronto Research Company, G797800); MitoPerox (Abcam, ab146820); MitoTracker Far Red (Thermo Fisher Scientific, M22426 ); MitoTracker Green (Thermo Fisher Scientific, M7514); SE Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XC-1024); P3 Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XP-3024); penicillin and streptomycin (Sigma-Aldrich, P0781); Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106×4); Pierce Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific, 88266); potassium chloride (Janssen Chimica, 7447407); protease inhibitor (Thermo Fisher Scientific, A32953); saponin (Sigma-Aldrich, 8047152); Gibco, Roswell Park Memorial Institute Sodium Chloride, (Sigma-Aldrich, A0431796); SYTOX Green (Sigma-Aldrich, S7020); TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR 6000); Tris base (Sigma-Aldrich, 77861); Triton (Sigma-Aldrich, T9234); Tween (Sigma-Aldrich, P4780).

Techniques: Transfection, Marker, Staining

Journal: Nature Cell Biology

Article Title: Endoplasmic reticulum–mitochondria contacts are prime hotspots of phospholipid peroxidation driving ferroptosis

doi: 10.1038/s41556-025-01668-z

Figure Lengend Snippet: a , Representative immunoblot for PERK in PERK WT and PERK KO MEFs. ACTIN serves as loading control. Arrow indicates PERK. b , Representative TEM images of PERK WT and PERK KO MEFs transiently transfected with HRP-KDEL. c , quantification of the EMCSs N normalized on the mitochondrial surface in PERK WT and PERK KO MEFs. d , Representative FRET (Fluorescence Resonance Energy Transfer) live microscopy images of PERK WT MEFs transiently transfected with AKAP1-mRFP control or 9xL spacer and infected with FEMP probe (mAKAP1-YFP-Tav2-CFP-Sac1) in basal condition. Scale bar, 10 µm. Zoom, 10 µm. e , FRET-based ER-Mitochondria Proximity (FEMP) ratio in MEFs transiently transfected with AKAP1-mRFP control or 9xL spacer and transiently infected with FEMP probe and treated with RSL3 (0.5 µM). (n = 3 biological replicates, multiple areas in the same well were acquired and analyzed per each timepoint per condition per biological replicate). f , Total Cell Liperfluo intensity normalized on total cellular volume (MFI) at time 0 and 5, 15, 30 ‘after RSL3 (0.5 µM) treatment in MEFs transiently transfected with AKAP1-mRFP control or 9xL spacer. (n = 3 biological replicates). g , Relative levels of total PLs in PERK KO MEFs (to PERK WT ). (n = 3 biological replicates). h , Relative levels of total PLs in MEFs transiently transfected with 9xL spacer (to AKAP1-mRFP control). (n = 3 biological replicates). All quantitative data are mean ± SEM. In c, e , statistical significance was determined by two sided unpaired t-test. In f , statistical significance was determined by two-way RM ANOVA, Tukey post-hoc test. In g, h , statistical significance was determined by one sample t-test. NS, not significant (P > 0.05), *P ≤ 0.05, **P < 0.01, ***P < 0.001. Source numerical and statistical data are provided.

Article Snippet: The reagents used were: Bodipy 581/591 C11 (Thermo Fisher Scientific); Liperfluo (Dojindo), d -glucose (Sigma-Aldrich, G7021-1KG); DAPI (Thermo Fisher Scientific, 62248); DAPI (Thermo Fisher Scientific, D1306); DMEM—high glucose (Sigma-Aldrich, D0422); Gibco DMEM/F-12 (Thermo Fisher Scientific, 11320074); Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich, DUO82040); GSK2606414 PERK Inhibitor (Toronto Research Company, G797800); MitoPerox (Abcam, ab146820); MitoTracker Far Red (Thermo Fisher Scientific, M22426 ); MitoTracker Green (Thermo Fisher Scientific, M7514); SE Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XC-1024); P3 Cell Line 4D-NucleofectorTM X Kit L (Lonza Bioscience, V4XP-3024); penicillin and streptomycin (Sigma-Aldrich, P0781); Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106×4); Pierce Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific, 88266); potassium chloride (Janssen Chimica, 7447407); protease inhibitor (Thermo Fisher Scientific, A32953); saponin (Sigma-Aldrich, 8047152); Gibco, Roswell Park Memorial Institute Sodium Chloride, (Sigma-Aldrich, A0431796); SYTOX Green (Sigma-Aldrich, S7020); TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR 6000); Tris base (Sigma-Aldrich, 77861); Triton (Sigma-Aldrich, T9234); Tween (Sigma-Aldrich, P4780).

Techniques: Western Blot, Control, Transfection, Fluorescence, Förster Resonance Energy Transfer, Microscopy, Infection