Fig. 5. LiZIP3 expression in Leishmania is modulated by the concentration of zinc in the culture medium.

A. Dose-dependent effect of EDTA on LiZIP3 levels. The effect of the divalent metal chelator on the expression levels of LiZIP3 was addressed by adding increasing concentrations of EDTA to the culture medium for 48 h. Analysis was performed by western blotting. Values below the blots refer to the densitometry analysis (the intensity of the LiZIP3 band in the presence of each EDTA concentration was compared to that obtained in the absence of the chelator after loading correction with LiGAPDH.

B and C. The presence of zinc in the culture media reduces LiZIP3 expression. The ability of zinc to decrease the LiZIP3 levels in the presence of chelating agents was assessed (B) by supplementing a culture of parasites grown for 24 h in 0.4 mM EDTA with 50 μM zinc or (C) by adding 10 μM TPEN or 10 μM TPEN plus 50 μM zinc to a culture of parasites previously grown for 48 h in normal medium. Twenty four hours upon medium supplementation, parasites were harvested and processed for western blotting.

D. Effect of different divalent metals on LiZIP3 expression. L. infantum promastigotes were inoculated in normal medium at 10⁶ mL⁻¹ and, at 48 h of growth, several divalent metals were added to the cultures for an additional 24 h. The effect of metal supplementation on the level of LiZIP3 expression was analysed by western blotting. Expression of LiGAPDH in the same blots was used as loading control in all cases.