Effect of ZnT8 down-regulation upon intracellular zinc levels as assessed via zinquin fluorescence.

<p>In panel A, untransfected INS-1 cells in 96-well microtiter plates were treated for 3 h with control (nominally zinc-free) medium and Chelex resin-treated fetal bovine serum (10% vol∶vol), or the identical medium/serum combination supplemented with zinc (75 µM zinc sulfate). Cells were then washed, incubated for 30 min in HBSS with the indicated concentration of zinquin ester, and washed again. Each bar represents mean±SEM of twelve replicates; depicted figure is representative of two separate experiments. Fluorescence was measured with excitation at 370 nm and emission at 490 nm. Exogenous zinc markedly increases zinquin fluorescence at all tested concentrations of the dye. In panel B, this microtiter plate-based zinquin fluorescence assay was applied to stable cells expressing VEC-shRNA or ZnT8-shRNA, following incubation with control medium (Zn 0) or with medium supplemented with 75 µM Zn (Zn 75 µM) or Ni (negative control; Ni 75 µM). Fluorescence under control conditions in the absence of zinquin is also shown (No Dye). Each bar represents mean±SEM of three separate experiments (i.e., three sets of stable transfectants, each with twelve replicates). * denotes <i>p</i><0.05 relative to the same experimental condition in VEC-shRNA stable transfectants. Down-regulation of ZnT8 resulted in less zinquin fluorescence in the absence (Zn 0) and presence (Zn 75 µM) of supplemental zinc; there was no effect upon fluorescence in the presence of nickel (Ni 75 µM) or in the absence of dye (No Dye).