Fig. 6.

Overexpression of GFP-tagged ZnT2 restores chelatable zinc stores in AP-3-depleted cells. (a-c) Cultured M1 fibroblasts were treated with control siRNA or with a siRNA duplex specifically designed to target the δ subunit of the AP-3 protein complex. (a) Whole-cell lysates were obtained and analyzed by immunoblotting, using antibodies against theβ3A and μ3A subunits of AP-3 as well as toα-tubulin andβ-actin. (b,c) M1 cells treated with siRNA were transiently transfected to overexpress GFP-ZnT2 or Znt3-GFP, and 24 h later they were sequentially incubated with ZnSO₄ and zinquin and analyzed by two-photon laser scanning microscopy. (b) GFP fluorescence levels in cells treated with siRNA specific to AP-3 δ and expressing either GFP-Znt2 or Znt3-GFP. Bars represent means ± SEM of values expressed in arbitrary units. NS, not significant (Student’s t-test). (c) Quantitative analysis of the levels of zinquin fluorescence observed in M1 fibroblasts treated with a control siRNA or with siRNA specific to AP-3 δ, and transiently transfected to overexpress GFP-tagged ZnT2 or ZnT3. The levels of zinquin fluorescence per cell were estimated by image analysis of 7-80 randomly selected cells per sample, and expressed as means ± SEM of the values normalized to the average zinquin fluorescence intensity of control cells. Kruskal-Wallis test followed by Dunn’s multiple comparison test: NS, not significant; ** p < 0.01.