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3d Reconstructions Volumetric Surface Reconstructions, supplied by Oxford Instruments, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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MtDNA release mediated by mPTP promotes ferroptosis. (A) Relative cytosolic mtDNA amounts <t>in</t> <t>RSL3‐treated</t> NCI‐H1299 WT and CypD‐KO cells. The relative ratios of ND1 mtDNA, 18S nuclear DNA (left panel), and actin (right panel) are shown. (B) Relative amounts of Ox‐mtDNA in cytosols of RSL3‐primed WT or CypD‐KO NCI‐H1299 cells. (C) Representative <t>3D</t> reconstruction images showing mitochondria (red, MitoTracker) and mitochondrial DNA (green, PicoGreen) in cells treated with DMSO or RSL3. Super‐resolution images were acquired using structured illumination microscopy (SIM). Mitochondria structures were labeled in red, and mtDNA signals were labeled in green (left panels). The mtDNA signals that were in contact with the mitochondrial surface were labeled in light pink, while those not in contact with the mitochondrial surface were in green (right panels, shown in the overlap images). Scale bar was 2 µm. (D) Quantification of cytosolic mtDNA (mtDNA in cytosol) in indicated groups. (E, F) ANT2 (E) and FEN1 (F) were knocked down by specific siRNA in NCI‐H1299 cells, followed by RSL3 treatment for 6 h to detect relative cytosolic mtDNA. (G) Western Blot analysis of FEN1 knockdown cells. (H) FEN1 was knocked down by specific siRNA in NCI‐H1299 cells, followed by indicated doses of erastin for 24 h to detect cell death. (I) NCI‐H1299 cells were transfected with siRNA specifically against FEN1 for 48 h, then subjected to 5 µ m erastin for 18 h. Lipid peroxidation was measured with C11‐BODIPY staining by flow cytometry. (J) Quantitative analysis of the fold change of lipid oxidation ratio in (I). The statistical significance between different groups (A, D, E, and F) was analyzed by One‐way ANOVA (Prism; GraphPad). Two‐Way ANOVA was used for (B and J).
3d Surface Reconstructions, supplied by Oxford Instruments, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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MtDNA release mediated by mPTP promotes ferroptosis. (A) Relative cytosolic mtDNA amounts <t>in</t> <t>RSL3‐treated</t> NCI‐H1299 WT and CypD‐KO cells. The relative ratios of ND1 mtDNA, 18S nuclear DNA (left panel), and actin (right panel) are shown. (B) Relative amounts of Ox‐mtDNA in cytosols of RSL3‐primed WT or CypD‐KO NCI‐H1299 cells. (C) Representative <t>3D</t> reconstruction images showing mitochondria (red, MitoTracker) and mitochondrial DNA (green, PicoGreen) in cells treated with DMSO or RSL3. Super‐resolution images were acquired using structured illumination microscopy (SIM). Mitochondria structures were labeled in red, and mtDNA signals were labeled in green (left panels). The mtDNA signals that were in contact with the mitochondrial surface were labeled in light pink, while those not in contact with the mitochondrial surface were in green (right panels, shown in the overlap images). Scale bar was 2 µm. (D) Quantification of cytosolic mtDNA (mtDNA in cytosol) in indicated groups. (E, F) ANT2 (E) and FEN1 (F) were knocked down by specific siRNA in NCI‐H1299 cells, followed by RSL3 treatment for 6 h to detect relative cytosolic mtDNA. (G) Western Blot analysis of FEN1 knockdown cells. (H) FEN1 was knocked down by specific siRNA in NCI‐H1299 cells, followed by indicated doses of erastin for 24 h to detect cell death. (I) NCI‐H1299 cells were transfected with siRNA specifically against FEN1 for 48 h, then subjected to 5 µ m erastin for 18 h. Lipid peroxidation was measured with C11‐BODIPY staining by flow cytometry. (J) Quantitative analysis of the fold change of lipid oxidation ratio in (I). The statistical significance between different groups (A, D, E, and F) was analyzed by One‐way ANOVA (Prism; GraphPad). Two‐Way ANOVA was used for (B and J).
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MtDNA release mediated by mPTP promotes ferroptosis. (A) Relative cytosolic mtDNA amounts <t>in</t> <t>RSL3‐treated</t> NCI‐H1299 WT and CypD‐KO cells. The relative ratios of ND1 mtDNA, 18S nuclear DNA (left panel), and actin (right panel) are shown. (B) Relative amounts of Ox‐mtDNA in cytosols of RSL3‐primed WT or CypD‐KO NCI‐H1299 cells. (C) Representative <t>3D</t> reconstruction images showing mitochondria (red, MitoTracker) and mitochondrial DNA (green, PicoGreen) in cells treated with DMSO or RSL3. Super‐resolution images were acquired using structured illumination microscopy (SIM). Mitochondria structures were labeled in red, and mtDNA signals were labeled in green (left panels). The mtDNA signals that were in contact with the mitochondrial surface were labeled in light pink, while those not in contact with the mitochondrial surface were in green (right panels, shown in the overlap images). Scale bar was 2 µm. (D) Quantification of cytosolic mtDNA (mtDNA in cytosol) in indicated groups. (E, F) ANT2 (E) and FEN1 (F) were knocked down by specific siRNA in NCI‐H1299 cells, followed by RSL3 treatment for 6 h to detect relative cytosolic mtDNA. (G) Western Blot analysis of FEN1 knockdown cells. (H) FEN1 was knocked down by specific siRNA in NCI‐H1299 cells, followed by indicated doses of erastin for 24 h to detect cell death. (I) NCI‐H1299 cells were transfected with siRNA specifically against FEN1 for 48 h, then subjected to 5 µ m erastin for 18 h. Lipid peroxidation was measured with C11‐BODIPY staining by flow cytometry. (J) Quantitative analysis of the fold change of lipid oxidation ratio in (I). The statistical significance between different groups (A, D, E, and F) was analyzed by One‐way ANOVA (Prism; GraphPad). Two‐Way ANOVA was used for (B and J).
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MtDNA release mediated by mPTP promotes ferroptosis. (A) Relative cytosolic mtDNA amounts <t>in</t> <t>RSL3‐treated</t> NCI‐H1299 WT and CypD‐KO cells. The relative ratios of ND1 mtDNA, 18S nuclear DNA (left panel), and actin (right panel) are shown. (B) Relative amounts of Ox‐mtDNA in cytosols of RSL3‐primed WT or CypD‐KO NCI‐H1299 cells. (C) Representative <t>3D</t> reconstruction images showing mitochondria (red, MitoTracker) and mitochondrial DNA (green, PicoGreen) in cells treated with DMSO or RSL3. Super‐resolution images were acquired using structured illumination microscopy (SIM). Mitochondria structures were labeled in red, and mtDNA signals were labeled in green (left panels). The mtDNA signals that were in contact with the mitochondrial surface were labeled in light pink, while those not in contact with the mitochondrial surface were in green (right panels, shown in the overlap images). Scale bar was 2 µm. (D) Quantification of cytosolic mtDNA (mtDNA in cytosol) in indicated groups. (E, F) ANT2 (E) and FEN1 (F) were knocked down by specific siRNA in NCI‐H1299 cells, followed by RSL3 treatment for 6 h to detect relative cytosolic mtDNA. (G) Western Blot analysis of FEN1 knockdown cells. (H) FEN1 was knocked down by specific siRNA in NCI‐H1299 cells, followed by indicated doses of erastin for 24 h to detect cell death. (I) NCI‐H1299 cells were transfected with siRNA specifically against FEN1 for 48 h, then subjected to 5 µ m erastin for 18 h. Lipid peroxidation was measured with C11‐BODIPY staining by flow cytometry. (J) Quantitative analysis of the fold change of lipid oxidation ratio in (I). The statistical significance between different groups (A, D, E, and F) was analyzed by One‐way ANOVA (Prism; GraphPad). Two‐Way ANOVA was used for (B and J).
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MtDNA release mediated by mPTP promotes ferroptosis. (A) Relative cytosolic mtDNA amounts in RSL3‐treated NCI‐H1299 WT and CypD‐KO cells. The relative ratios of ND1 mtDNA, 18S nuclear DNA (left panel), and actin (right panel) are shown. (B) Relative amounts of Ox‐mtDNA in cytosols of RSL3‐primed WT or CypD‐KO NCI‐H1299 cells. (C) Representative 3D reconstruction images showing mitochondria (red, MitoTracker) and mitochondrial DNA (green, PicoGreen) in cells treated with DMSO or RSL3. Super‐resolution images were acquired using structured illumination microscopy (SIM). Mitochondria structures were labeled in red, and mtDNA signals were labeled in green (left panels). The mtDNA signals that were in contact with the mitochondrial surface were labeled in light pink, while those not in contact with the mitochondrial surface were in green (right panels, shown in the overlap images). Scale bar was 2 µm. (D) Quantification of cytosolic mtDNA (mtDNA in cytosol) in indicated groups. (E, F) ANT2 (E) and FEN1 (F) were knocked down by specific siRNA in NCI‐H1299 cells, followed by RSL3 treatment for 6 h to detect relative cytosolic mtDNA. (G) Western Blot analysis of FEN1 knockdown cells. (H) FEN1 was knocked down by specific siRNA in NCI‐H1299 cells, followed by indicated doses of erastin for 24 h to detect cell death. (I) NCI‐H1299 cells were transfected with siRNA specifically against FEN1 for 48 h, then subjected to 5 µ m erastin for 18 h. Lipid peroxidation was measured with C11‐BODIPY staining by flow cytometry. (J) Quantitative analysis of the fold change of lipid oxidation ratio in (I). The statistical significance between different groups (A, D, E, and F) was analyzed by One‐way ANOVA (Prism; GraphPad). Two‐Way ANOVA was used for (B and J).

Journal: Advanced Science

Article Title: CypD Dependent mPTP Opening Is Crucial for Oxidized Mitochondrial DNA Release in Ferroptosis

doi: 10.1002/advs.202502239

Figure Lengend Snippet: MtDNA release mediated by mPTP promotes ferroptosis. (A) Relative cytosolic mtDNA amounts in RSL3‐treated NCI‐H1299 WT and CypD‐KO cells. The relative ratios of ND1 mtDNA, 18S nuclear DNA (left panel), and actin (right panel) are shown. (B) Relative amounts of Ox‐mtDNA in cytosols of RSL3‐primed WT or CypD‐KO NCI‐H1299 cells. (C) Representative 3D reconstruction images showing mitochondria (red, MitoTracker) and mitochondrial DNA (green, PicoGreen) in cells treated with DMSO or RSL3. Super‐resolution images were acquired using structured illumination microscopy (SIM). Mitochondria structures were labeled in red, and mtDNA signals were labeled in green (left panels). The mtDNA signals that were in contact with the mitochondrial surface were labeled in light pink, while those not in contact with the mitochondrial surface were in green (right panels, shown in the overlap images). Scale bar was 2 µm. (D) Quantification of cytosolic mtDNA (mtDNA in cytosol) in indicated groups. (E, F) ANT2 (E) and FEN1 (F) were knocked down by specific siRNA in NCI‐H1299 cells, followed by RSL3 treatment for 6 h to detect relative cytosolic mtDNA. (G) Western Blot analysis of FEN1 knockdown cells. (H) FEN1 was knocked down by specific siRNA in NCI‐H1299 cells, followed by indicated doses of erastin for 24 h to detect cell death. (I) NCI‐H1299 cells were transfected with siRNA specifically against FEN1 for 48 h, then subjected to 5 µ m erastin for 18 h. Lipid peroxidation was measured with C11‐BODIPY staining by flow cytometry. (J) Quantitative analysis of the fold change of lipid oxidation ratio in (I). The statistical significance between different groups (A, D, E, and F) was analyzed by One‐way ANOVA (Prism; GraphPad). Two‐Way ANOVA was used for (B and J).

Article Snippet: 3D surface reconstructions were generated with Imaris software, revealing that, upon RSL3 treatment, mtDNA signals were redistributed from mitochondria into the cytoplasm (Figure ).

Techniques: Microscopy, Labeling, Western Blot, Knockdown, Transfection, Staining, Flow Cytometry