Figure 3

(a) Box and whisker plot showing the distribution of mortality in percentage of ISE6 cells exposed to different concentrations of ferrous sulphate for 48 h. Two hundred and fifty microliters of 1 × 10⁶ cells/ml was transferred to a 48-well plate and incubated overnight. The next day, the media were replaced by ferrous sulphate–enriched media at different concentrations (0, 0.1, 1, and 2 mM). Mortality was checked after 48 h using Trypan blue stain. A one-way ANOVA with Bonferonni multiple comparison tests was performed (Tables S5 and S6). (b) FER1 mRNA expression of ferrous sulphate (0, 0.1, 1, and 2 mM)–exposed cells for FER1 was observed using RT-PCR. 16S rRNA was used as a loading control. Full length gels are presented in Fig. S5. The RT-PCR result is representative of three separate experiments. (c) FER1 protein expression of ferrous sulphate (0, 0.1, 1, and 2 mM)–exposed cells for FER1 was observed using Western blotting. Anti-mouse tubulin was used as a loading control. Full length blots are presented in Fig. S2. The Western blotting result is representative of three separate experiments. (d) Localization of FER1 protein was observed using IFAT. Arrowheads indicate positive fluorescence. The IFAT result is representative of three separate experiments. Bar = 10 μm. The data presented are results of three independent experiments with the Trypan blue staining performed in two technical replicates for each experiment.