Fig. 5: ZIP8 enhances Zn²⁺-mediated GPX4 protein levels to inhibit ferroptosis.

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 (Fe²⁺) 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, independent experiments). Data are represented as mean ± SD.