fig6

Figure 6. Zinc ions reduce cholesterol level. (A) RNA sequencing analysis was carried out on DEGs identified in both the zinc gluconate and ZnSO₄ models, then subjected to KEGG enrichment analysis of pathways associated with lipid metabolism; (B) Expression levels of gene clusters associated with cholesterol metabolism; (C) Expression of gene clusters associated with cholesterol synthase; (D) Huh7.5.1 cells incubated with 20 μmol/L ZnSO₄ for 17 h were labeled with filipin. Scale bar: 10 µm; (E) Statistical analysis of relative membrane cholesterol levels in ZnSO₄-treated cells. For each group, at least 220 cells were analyzed across three independent experiments; (F) Huh7.5.1 cells incubated with 20 μmol/L zinc gluconate for 17 h. were labeled with filipin. Scale bar: 10 µm; (G) Statistical analysis of relative membrane cholesterol levels in zinc gluconate-treated cells. For each group, at least 255 cells were analyzed across three independent experiments; (H) Images of eGFP-TSPAN4-expressing Huh7.5.1 cells incubated with 20 μmol/L zinc gluconate, 20 μmol/L ZnSO₄, zinc gluconate with cholesterol, ZnSO₄ with cholesterol, and 25 μmol/L cholesterol for 17 h. Scale bar: 10 µm; (I) Statistical analyses of migrasome counts per 10 μm retraction fibers in (H); (J) Statistical analyses of retraction fiber lengths in (H). For each group, at least 206 cells were analyzed across three independent experiments. Data are shown as mean ± SEM. ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001; (K) Zinc ions inhibit migrasome formation, whereas cholesterol supplementation partially restores it. DEGs: Differentially expressed genes; eGFP: enhanced green fluorescent protein; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; KEGG: Kyoto Encyclopedia of Genes and Genomes; SEM: standard error of the mean; TSPAN4: tetraspanin 4; TPM: transcripts per million.