Supplementary Figure 2: Mutant forms of ZIP7 are expressed but display reduced Zn transporter activity.

(a) Fluorescence micrographs revealing expression of endogenous ZIP7 in primary dermal fibroblasts from healthy control or P1, stained with antibody against ZIP7 (red) and DAPI (blue). Scale bar, 20µm. (b) Impaired Zn²⁺ conductance of mutant forms of ZIP7 expressed in Xenopus oocytes, visualized by zinquin fluorescence. Left, fluorescence micrographs showing Zn²⁺-related zinquin signal and right, pairwise image analysis in ImageJ, as described in Methods. Images are representative of 4 independent experiments as exemplified in Fig 2h. (c) Western blot of detergent extracts of Xenopus oocytes injected in parallel with those visualized in (b), revealing expression of the recombinant ZIP7 proteins. Images are representative of 3 independent experiments. (d-e) Cytoplasmic Zn²⁺ concentration in HEK-293T cells stably expressing the genetically encoded cytoplasmic Zn²⁺ sensor eCALWY-4 and transfected with empty vector (EV), or vectors encoding WT, E363K (d) or P190A (e) ZIP7 proteins. Cytoplasmic Zn²⁺ concentration was calculated by comparing the steady state live cell fluorescence intensity with maximum and minimum signals obtained in the presence of TPEN and zinc pyrithione respectively, as described in Methods. Total number of cells analyzed was 80 (EV), 55 (WT), 26 (E363K) and 42 (P190A) across 2–4 experiments. Columns show means and bars the standard error. Comparisons were by one-way ANOVA with Bonferroni’s correction; * indicates p=0.0258 and ** p=0.0051.