Figure 3.

Effects of NtBHA posttreatment on iron homeostasis and oxidative stress levels in RPE cells. A) NtBHA posttreatment resulted in a decrease in levels of total iron in RPE cells treated with FAC (250 µM, 4 days) as determined by ICP. RPE cells were treated with NtBHA (250 µM, 16 h) after FAC exposure, and demonstrated a significant decrease in the levels of total iron compared with RPE cells treated with medium alone (0 µM NtBHA). The data represent mean ± sd of 3 independent experiments (**P<0.01, ***P<0.001). B) Effect of iron overload and NtBHA posttreatment on the levels of labile iron in RPE cells. Different treatments are indicated as follows: control = untreated RPE cells; NtBHA = RPE cells treated with 250 µM NtBHA; FAC, 4 days = RPE cells treated with 250 µM FAC for 4 days; FAC + 0 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days and posttreated with NtBHA-free cell medium; FAC + 250 µM NtBHA = RPE cells treated with 250 µM FAC for 4 days and posttreated with 250 µM NtBHA. Changes in labile iron levels were measured with Fe-sensitive calcein dye. FAC treatment for 4 days promoted significant decrease in calcein fluorescence, indicating increased labile iron concentration. Treatment with 250 µM NtBHA of iron-loaded cells reduced the levels of intracellular labile iron compared with RPE cells treated with medium alone (0 µM NtBHA). The data represent mean ± sd of 7 samples from 4 independent experiments (***P<0.001). C) Effect of NtBHA posttreatment on the levels of intracellular oxidants in iron-treated RPE cells. RPE cells were treated overnight (16 h) with different concentrations of NtBHA after 4 days of FAC exposure. Changes in oxidant levels were measured with oxidant-sensitive dye H₂DCF. Data are expressed as mean ± sd of 3 independent experiments. Each experiment was performed in duplicate (**P<0.01, ***P<0.001). D) Fluorescein diacetate dye was used as a control to check for potential changes in RPE dye loading. Different treatments promoted no significant changes in fluorescein signal indicating no difference in loading differently treated RPE cells with dyes.