fig3

Figure 3. Preventing extracellular zinc ions entry can reverse migrasome inhibition. (A) Images of eGFP-TSPAN4-expressing Huh7.5.1 cells incubated with 20 μmol/L zinc gluconate, 10 ng/mL Anti-SLC39A6 (which inhibits zinc ion entry into cells), zinc gluconate with Anti-SLC39A6, or a mock treatment for 17 h and stained with TSQ for zinc ions. Scale bar: 40 µm; (B) Migrasome counts per 10 μm retraction fibers in (A). For each group, at least 123 cells were analyzed across three independent experiments; (C) Statistical analyses of relative level of cytoplasmic zinc ions in (A). For each group, at least 85 cells were analyzed across three independent experiments; (D) Images of eGFP-TSPAN4-expressing Huh7.5.1 cells incubated with 20 μmol/L ZnSO₄, 10 ng/mL Anti-SLC39A6, ZnSO₄ with Anti-SLC39A6, or mock treatment for 17 h and stained with TSQ for zinc ions. Scale bar: 40 µm; (E) Migrasome counts per 10 μm retraction fibers in (D). For each group, at least 108 cells were analyzed across three independent experiments; (F) Statistical analyses of relative level of cytoplasmic zinc ions in (D). For each group, at least 106 cells were analyzed across three independent experiments; (G) Concentration of zinc ions in the huh7.5.1 cell culture after treatment with 20 μmol/L ZnSO₄, 20 μmol/L zinc gluconate, 10 ng/mL Anti-SLC39A6, ZnSO₄ with Anti-SLC39A6, zinc gluconate with Anti-SLC39A6 and mock for 17 h. Data are shown as mean ± SEM. NS denotes P > 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001; (H) Schematic diagram illustrating how Anti-SLC39A6 blocks zinc ions entry into cells. eGFP: Enhanced green fluorescent protein; NS: not significant; SEM: standard error of the mean; SLC39A: solute carrier family 39A; SLC39A6: solute carrier family 39 member 6; TSPAN4: tetraspanin 4; TSQ: N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide.