Fig. 4.

Zn²⁺ accumulation in purified vesicles. (A and B) M7Vs isolated from SV40MES-13 cells were loaded with FluoZin-3-AM and imaged in medium containing EGTA (A) or 15 nM Zn²⁺ (B). (C and D) Vesicles were perfused first with 15 nM Zn²⁺ and then with 1.4 nM Zn²⁺ or 50 µM TPEN. The mean, SD, and ANOVA statistics from four experiments are shown. *P < 0.001 compared with initial fluorescence; **P < 0.001 after TPEN addition. (E and F) Zn²⁺ trapping in vesicles is not caused by fluorophore accumulation: Zn²⁺-loaded vesicles were incubated with FluoZin-3-AM without Zn²⁺; then extravesicular dye was washed off, and remaining vesicular fluorescence was reduced by Zn²⁺ chelation by membrane-permeant TPEN but not membrane-impermeant EGTA. Fluorescence intensities in F were normalized to those in TPEN. Results are shown as the mean and SD from three experiments are shown; *P < 0.05; ANOVA. See also Fig. S4 A and B.