Effects of nifedipine and photodegraded nifedipine on metal-ion transport in vitro. Data presented are mean and SD for n observations. We performed statistical analyses (1- or 2-way analysis of variance [ANOVA or 2ANOVA] and 2-tailed post-hoc all-pairwise comparisons using the Holm-Šidák test) using SigmaStat version 3.5 (Systat Software) with critical significance level α = 0.05. (A) Effects of freshly prepared nifedipine (Nifd) and photodegraded nifedipine (PDN) on uptake of 4μM ⁵⁹Fe²⁺ (n = 3) in HEK293 cells transiently transfected with human DMT1 1A/−IRE. 2ANOVA and post-hoc pairwise comparisons revealed no interaction (P = .65) and that neither Nifd (unadjusted P = .27) nor PDN (unadjusted P = .062) differed from untreated. (B-C) Effects of Nifd and PDN on iron transport in a regulated-expression system in HEK293 cells. Nontransfected cells contained only the tetracycline-responsive element (TetR); HEK293 cells were stably transfected with the empty vector (EV) or with rodent 1A/+IRE and 2/−IRE isoforms of rodent DMT1. Expression was induced by treatment with 25nM doxycycline (Doxy). Immunoblot in panel B was stained by an antibody that recognizes all DMT1 isoforms. Uptake of 2μM ⁵⁹Fe²⁺ (C; n = 2). 2ANOVA revealed an effect of PDN (P < .001), but not Nifd (P = .55), when [Doxy] = 0. When [Doxy] = 25nM, we found no treatment effects (P = .36). (D) Effect of Nifd and PDN on DMT1-mediated Fe²⁺-evoked currents. A typical continuous current record from an oocyte expressing DMT1 and voltage-clamped at −70 mV is displayed (corrected for baseline drift). The oocyte was superfused with pH 7.5 medium for the periods shown by the empty portions of top bar or pH 5.5 medium (cross-hatched boxes). We presented 50μM Fe²⁺ (■) alone or with 100μM Nifd (hatched box) or PDN (gray box). (E) Effect of Nifd on ⁵⁵Fe²⁺ transport at pH 6.5 (n = 94-97). 2ANOVA revealed no interaction (P = .89). (F) Effects of Nifd and PDN on ⁵⁵Fe²⁺ transport at pH 5.5 (n = 22-26). 2ANOVA revealed an interaction (P < .001); within DMT1, a indicates not significant (unadjusted P = .93) and b indicates unadjusted P < .001 compared with no treatment; within PDN, c indicates not significant (unadjusted P = .99) compared with control. (G) Effect of varying PDN-treatment time on ⁵⁵Fe²⁺ transport at pH 5.5 in control (noninjected) oocytes. ⁵⁵Fe²⁺ uptake was measured in untreated noninjected oocytes (None) and in noninjected oocytes that were coincubated with 100μM PDN after a preincubation period with 100μM PDN ranging from 0 to 60 minutes (n = 16-19). ANOVA (P < .001) and post-hoc pairwise comparisons indicated that all groups differed from one another (unadjusted P < .01) except 0 and 30 minutes (unadjusted P = .09). Materials and methods are described in supplemental information.