fig2

Figure 2. Removal of intracellular zinc ions can reverse the inhibition of migrasome biogenesis induced by zinc ions. (A) Images of eGFP-TSPAN4-expressing Huh7.5.1 cells incubated with 20 μmol/L zinc gluconate, 5 μmol/L disulfiram (which can pump zinc ions out of the cells), zinc gluconate with disulfiram, or a mock treatment for 17 h and stained with TSQ for zinc. Scale bar: 40 µm; (B) Statistical analyses of migrasome counts per 10 μm retraction fibers in (A). For each group, at least 122 cells were analyzed across three independent experiments; (C) Statistical analyses of relative level of cytoplasmic zinc in (A). For each group, at least 135 cells were analyzed across three independent experiments; (D) Images of eGFP-TSPAN4-expressing Huh7.5.1 cells incubated with 20 μmol/L ZnSO₄, disulfiram, ZnSO₄ with disulfiram, or mock treatment for 17 h and stained with TSQ. Scale bar: 40 µm; (E) Migrasome counts per 10 μm retraction fibers from (D). For each group, at least 111 cells were analyzed across three independent experiments; (F) Statistical analyses of relative level of cytoplasmic zinc in (D). For each group, at least 109 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, 5 μmol/L disulfiram, ZnSO₄ with disulfiram, zinc gluconate with disulfiram and mock for 17 h. Data are shown as mean ± SEM. NS denotes P > 0.05; ^***P < 0.001; ^****P < 0.0001; (H) Diagram of disulfiram’s role in expelling intracellular zinc ions to the extracellular space. eGFP: Enhanced green fluorescent protein; NS: not significant; SEM: standard error of the mean; SLC30A: solute carrier family 30A; SLC39A: solute carrier family 39A; TSPAN4: tetraspanin 4; TSQ: N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide.