RPA staining of live zebrafish embryos confirms decrease of mitochondrial iron in erythroid cells of mutant zebrafish lines and ability of Fe-hinokitiol to increase erythroid mitochondrial Fe²⁺ levels in mutant fish. (A) frs/frs mutant lcr:GFP⁺ erythroid cells have an increase in RPA signal, indicating significantly decreased Fe²⁺ levels relative to WT cells. Addition of iron-hinokitiol decreased RPA signal, indicating increased mitochondrial Fe²⁺. (B) frs/frs mutant lcr:GFP^– (nonerythroid) cells did not significantly differ from WT cells in their RPA signal, indicating that Mfrn1 was largely not required for maintenance of nonerythroid mitochondrial Fe²⁺ levels. Addition of supplemental iron did not alter Fe²⁺ levels in frs/frs nonerythroid cells. (C) weh/weh mutant lcr:GFP⁺ erythroid cells have increased RPA signal, indicating significantly decreased Fe²⁺ levels relative to weh/+ cells. Addition of iron-hinokitiol decreased RPA signal, indicating increased mitochondrial Fe²⁺. (D) weh/weh mutant lcr:GFP^– (nonerythroid) cells did not significantly differ from WT cells in their RPA signal, indicating that Fpn1 (despite its requirement for embryonic use of yolk iron) was largely not required for maintenance of nonerythroid mitochondrial Fe²⁺ levels. Addition of supplemental iron did not alter Fe²⁺ levels in weh/weh nonerythroid cells. ∗∗∗ Indicates statistical significance at 95% significance. Dashed red lines indicate 90% confidence intervals as defined by WT controls; red data points indicate data points that fall outside these 90% confidence intervals.