Fig. 2

Subcellular localisation of hZip1 protein in Caco-2 cells was not altered by zinc levels but differences in the expression of endogenous hZip1 mRNA were detected in response to changes in zinc treatment and differentiation. A Caco-2 cells were grown in control medium (b), Zn-supplemented (c and d) or Zn-deficient (e and f) media for 48 h and incubated with hZip1 Ab. Cells were stained with hZip1 antibody with fourfold excess of antigen to test the Ab specificity (a). Untreated medium (b), medium supplemented with 100 μM zinc (c), medium with 100 μM zinc and 0.2 nM pyrithione (d) zinc-deficient medium with 6 μM TPEN (e) and also Chelex-treated medium (f). The XZ sections (b’–f’). B Real-time PCR analysis showed down-regulation on hZip1 mRNA in Caco-2 cells exposed to 100 μM zinc (grey bar) and 100 μM zinc with 0.2 nM pyrithione (blue bar), while 6 μM TPEN treatment (light green bar) and incubation with Chelex-treated media (dark green bar) resulted in increased levels of hZIP1 transcript. In differentiated Caco-2 up-regulation, the hZip1 levels were detected (dark grey bar) compared to the control (solid white bar). The bars represent the mean (±SD) of 3 independent experiments. Stars indicate the statistically significant differences (p < 0.05, t test)