Journal: Cell Death & Disease

Article Title: ZIP8 modulates ferroptosis to drive esophageal carcinoma progression

doi: 10.1038/s41419-025-07692-z

Figure Lengend Snippet: A Functional schematic illustrating the role of ZIP8 in the ferroptosis pathway. B Sensitivity of ESCC cells to the ferroptosis activator Erastin, assessed by CCK-8 assay following treatment with increasing concentrations of Erastin (0 µM, 0.01 µM, 0.1 µM, 1 µM, 10 µM). Data represent mean ± SD of n = 3 independent experiments. C Lipid peroxidation assay to evaluate oxidative damage, with malondialdehyde (MDA) concentration measured using an MDA assay kit. D Western blot analysis of FTL and FTH1 expression in ESCC cells following ZIP8 knockdown. E , F Detection of lipid peroxidation in ZIP8 knockdown cells using the C11-BODIPY fluorescent probe. Scale bar, 20 µm. G , H Assessment of lipid peroxidation in KYSE30 and KYSE510 cells using the BD FACSAria II flow cytometer with the BODIPY™ 581/591 C11 probe. I , J Measurement of intracellular ferrous iron (Fe 2+ ) concentration in ZIP8 knockdown cells using the FerroOrange fluorescent probe. Scale bar, 20 µm. Student’s unpaired t-test was employed for statistical analysis in ( B , C , F , H , J ). * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 3, independent experiments). Data are represented as mean ± SD.

Article Snippet: G , H Assessment of lipid peroxidation in KYSE30 and KYSE510 cells using the BD FACSAria II flow cytometer with the BODIPYTM 581/591 C11 probe.

Techniques: Functional Assay, CCK-8 Assay, Peroxidation Assay, Concentration Assay, Multiple Displacement Amplification, Western Blot, Expressing, Knockdown, Flow Cytometry

Journal: Cell Death & Disease

Article Title: ZIP8 modulates ferroptosis to drive esophageal carcinoma progression

doi: 10.1038/s41419-025-07692-z

Figure Lengend Snippet: A , B Detection of intracellular zinc ion concentration in ZIP8-knockdown ESCC cells using the fluorescent probe Zinpyr-1. Scale bar, 20 µm. C Western blot analysis showing the expression of GPX4 in ZIP8 knock-down ESCC cells. D Changes in lipid peroxidation in esophageal cancer cells KYSE30 under zinc deficiency and overload conditions. Upper: Zinc deficiency-induced lipid peroxidation using 10 µM TPEN for 2 h. Lower: Zinc overload-modulated lipid peroxidation using 10 µM zinc sulfate for 5 h in ZIP8 knockdown cells. Immunofluorescence images were captured using a confocal microscope with BODIPY™ 581/591 C11 probe. Scale bar, 20 µm. E Immunofluorescence analyzed the GPX4 expression level in zinc deficiency and overload conditions in esophageal cancer cells KYSE30. Upper: Zinc deficiency-induced GPX4 changes using 10 µM TPEN for 2 h. Lower: Zinc overload-modulated GPX4 changes using 0.5 mM zinc sulfate for 5 h in ZIP8 knockdown cells. Scale bar, 20 µm. F Western blot showing changes in ZIP8-overexpressing ESCC cells with the CREB inhibitor 666-15. G Sensitivity of ZIP8-overexpressing ESCC cells with the CREB inhibitor 666-15 to the ferroptosis activator Erastin. Cell viability was assessed by CCK-8 assay. H Lipid peroxidation analysis of ESCC after treating with the CREB inhibitor under ZIP8 upregulation. I , J Measurement of intracellular ferrous iron (Fe 2+ ) concentration using FerroOrange in cells overexpressing ZIP8 with the CREB inhibitor 666-15. Scale bar, 20 µm. K , L Assessment of lipid peroxidation in ZIP8 overexpressed or 666-15 treated KYSE30 and KYSE450 cells. Cells were treated with the CREB inhibitor 666-15 at a concentration of 5 µM for 5 h and assessed using the BD FACSAria II flow cytometer with the BODIPY™ 581/591 C11 probe. Statistical analysis was performed using Student’s unpaired t test ( B , D , E , G , H , J , L ). * p < 0.05, ** p < 0.01, *** p < 0.001 ( n = 3, inde p endent experiments). Data are represented as mean ± SD.

Article Snippet: G , H Assessment of lipid peroxidation in KYSE30 and KYSE510 cells using the BD FACSAria II flow cytometer with the BODIPYTM 581/591 C11 probe.

Techniques: Concentration Assay, Knockdown, Western Blot, Expressing, Immunofluorescence, Microscopy, CCK-8 Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: ZIP8 modulates ferroptosis to drive esophageal carcinoma progression

doi: 10.1038/s41419-025-07692-z

Figure Lengend Snippet: A CCK-8 assay showing the effect of Nobiletin treatment on ESCC cells pretreated with DMSO and various inhibitors (Fer-1, Z-VAD-FMK, 3-MA) for 24 h. B Lipid peroxidation levels, indicated by malondialdehyde (MDA) concentration, measured using an MDA assay kit after treatment with different concentrations of Nobiletin (0 μM, 10 μM, 20 μM, 40 μM). C Western blot analysis of FTL and FTH1 expression in ESCC cells treated with increasing concentrations of Nobiletin (0 μM, 10 μM, 20 μM, 40 μM). D Cells were treated with DMSO or Nobiletin (40 μM), followed by the addition of increasing concentrations of erastin (0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM). Cell proliferation was assessed using the CCK-8 assay. E Intracellular zinc ion concentration in ESCC cells treated with Nobiletin, measured using the fluorescent probe Zinpyr-1. Scale bar, 20 μm. F Western blot analysis of p-CREB, CREB, and GPX4 expression in ESCC cells treated with different concentrations of Nobiletin. G Intracellular ferrous iron (Fe 2+ ) concentration measured using FerroOrange in cells treated with different concentrations of Nobiletin. Scale bar, 20 μm. H Lipid peroxidation levels measured using the fluorescent probe C11-BODIPY in cells treated with different concentrations of Nobiletin. Scale bar, 20 μm. I Assessment of lipid peroxidation in KYSE30 and KYSE510 cells using the BD FACSAria II flow cytometer with the BODIPY™ 581/591 C11 probe after treatment with Nobiletin for 24 h. J Cell proliferation was assessed using the CCK-8 assay following treatment with ferrostatin-1 (Fer-1) in Nobiletin-treated or ZIP8 knockdown cells. n = 3, independent experiments. Student’s unpaired t test was used for statistical analysis ( A , B , D , E , G , H , I , J). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are re p resented as mean ± SD.

Article Snippet: G , H Assessment of lipid peroxidation in KYSE30 and KYSE510 cells using the BD FACSAria II flow cytometer with the BODIPYTM 581/591 C11 probe.

Techniques: CCK-8 Assay, Concentration Assay, Multiple Displacement Amplification, Western Blot, Expressing, Flow Cytometry, Knockdown