dZip1 or dZip2 overexpression causes zinc accumulation. (A) Fluorescence of MtnB-eYFP was enhanced in the midgut constriction of the larvae overexpressing dZip1. Genotypes of the flies are MtnB-EYFP/+; da-GAL4/+ for the control larvae, and MtnB-EYFP/+; da-GAL4/UAS-dZip1 for the larvae overexpressing dZip1. Arrowheads denote the midgut constriction. Scale bars = 100 μm. (B) Reverse transcriptase (RT)-PCR analysis showing upregulation of MtnB and MtnC in larvae overexpressing dZip1. rp49 was used as the reference gene for normalization. (C) RT-PCR analysis of MT2a transcriptional induction by overexpression of dZip2 in Caco-2 cells. GAPDH was used as the reference gene for normalization. (D) Zinpyr-1 staining reveals intracellular zinc accumulation after dZip1 or dZip2 expression in Chinese hamster ovary (CHO) cells. Scale bars = 10 μm. (E) Ubiquitous overexpression of dZip1 specifically resulted in zinc sensitivity. (F) Gut-specific overexpression of dZip2 led to zinc sensitivity. Genotypes of flies are da-GAL4/+ for the control, da-GAL4/UAS-dZip1 for dZip1 overexpression; NP3084/+ for the gut-specific control fly) and NP3084/UAS-dZip2 for the gut-specific dZip2 overexpression fly. Values are presented as means ± SEM; n ≥ 3. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA.