Fig. 6: Nobiletin is a potent ZIP8 inhibitor.

A Analysis of the cancer HSP database to identify potential interactions between ZIP8 and traditional chinese medicine compounds. B Computational modeling depicting the binding interface between Nobiletin and ZIP8. C ZIP8 knockdown cell lines KYSE30, KYSE450, and KYSE510 were treated with increasing concentrations of Nobiletin (0 μM, 10 μM, 20 μM, 40 μM) to assess cell proliferation using the CCK-8 assay. Statistical analysis was performed using Student’s unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3, independent experiments). Data are represented as mean ± SD. D Drug affinity responsive target stability (DARTS) assay for ZIP8 identification in KYSE450 cells. E Solvent-induced protein precipitation (SIP) assay to assess the binding affinity of Nobiletin to ZIP8 in KYSE450 cells. F CETSA assay showing the binding affinity of Nobiletin to ZIP8 in KYSE450 cells. G Schematic diagram outlining the process of quantitative proteomics analysis. KYSE450 cells were treated with DMSO or Nobiletin (40 µM) for 24 h, followed by LC-MS/MS analysis. H Distribution of peptide length for the 7005 proteins identified. Peptide length is plotted on the X axis, and the number of peptides on the Y axis. I Volcano plot illustrating differential protein expression. J Heatmap representation of differentially expressed proteins between DMSO and Nobiletin (40 µM) groups. K Wikipathway enrichment analysis of differentially expressed proteins. L GSEA analysis of ZIP8’s role in ferroptosis based on proteomic analysis. M Sub-cellular localization of differentially expressed proteins.