Fig. 4: The zinc-ATF4-ARX axis mediates SC-β cell identity loss.

a Reactome pathway enrichment analysis of DEGs in SC-β cells of SC-islets treated with or without excessive zinc under high glucose using GSEA. b Panel showing expression changes of top genes ranked by significance in subcluster 3 of SC-β cells. c Violin plots showing ATF4 expression levels in subcluster 3 of SC-β cells. d qPCR analysis showing expression changes of ISR-related genes in SC-islets treated with or without ZnPTO. n = 3. e Western blot analysis of eIF2α, phosphorylated eIF2α, PERK, phosphorylated PERK, and ATF4 expression in SC-islets treated with or without ZnPTO. n = 3. Grayscale analysis of the blots is shown in Supplementary information, Fig. S16a. f ATF4 motif analyzed by JASPAR and putative ATF4-binding sites in the proximal promoter region of ARX. g Dual-luciferase reporter assays measuring relative ARX promoter activity using ARX-WT and ARX-mutation (Mut) constructs in MIN6 cells, with or without ATF4 OE. n = 7. h Dual-luciferase reporter assays measuring relative ARX promoter activity using ARX-WT constructs in MIN6 cells with ATF4 OE, treated with or without ZnPTO. n = 9. i Enrichment of ATF4 antibody-bound DNA was measured by qPCR after ChIP assays in SC-islets treated with or without ZnPTO. n = 10. j–l Representative immunofluorescent images (j) and quantification (k, l) of the proportion of INS-GFP⁺ARX⁺ cells among total INS-GFP⁺ cells, as well as the percentages of ARX⁺ cells (red) and INS-GFP⁺ cells (green) among the total number of DAPI⁺ cells (blue) in SC-islets infected with adenovirus carrying either an empty vector (n = 15) or an ATF4 OE construct (n = 17). Scale bar, 50 μm. m, n Representative immunofluorescent images (m) and quantification (n) of the percentages of ARX⁺ cells (red) among the total number of DAPI⁺ cells (blue) and INS-GFP ⁺ARX⁺ cells among total INS-GFP⁺ cells in SC-islets treated with (n = 9) or without ZnPTO (n = 10). Scale bar, 50 μm. o, p Representative immunofluorescent images (o) and quantification (p) of adherent SC-islets under control conditions (n = 17), ZnPTO treatment (n = 12), and ZnPTO treatment with 7 μM trans-ISRIB (n = 12). Scale bar, 25 μm. Two-way ANOVA with Sidak’s multiple-comparisons test was used to analyze data in g, i. One-way ANOVA with Dunnett’s multiple-comparisons test was used to analyze data in p. Unpaired two-tailed t-test was used to analyze data in d, h, k, l, n. ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. Individual data points are shown for all bar graphs.