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[Preprint]. 2024 Aug 9:2024.08.08.607074. [Version 1] doi: 10.1101/2024.08.08.607074

Figure 3: Iron toxicity modulates mitochondrial redox environment.

Figure 3:

A). Schematic diagram showing specific target of mitoTempo in redox environment. B). MitoTempo ameliorates iron toxicity induced increase in worm paralysis. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 10 μM MitoTempo, 35 μM iron and 35 μM iron + 10 μM MitoTempo. Paralysis was scored every 24 h for 5 days. Data are mean ±SEM, N=5 independent biological replicates (where one biological replicate contains 20 worms per plate). ***p=0.0001, ****p<0.0001, one-way ANOVA, Tukey post hoc test. C). MitoTempo restored non-paralyzed worm swimming rates in iron toxicity environment. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 10 μM MitoTempo, 35 μM iron and 35 μM iron + 10 μM MitoTempo. Non-paralyzed worms were individually transferred to plate containing 100 μl of buffer. After 30 seconds of equilibration, swimming rates were collected for 15 seconds. Data are mean ±SEM N=5 independent replicates (where 4 independent worm count constitute an N). ns not significant, **p<0.001, ****p<0.0001, one-way ANOVA, Tukey post hoc test. D). Schematic diagram showing specific target of SOD mimetic manganese porphyrin [Mn(III)PyP] in redox environment. E). Mn(III)PyP exacerbates iron toxicity induced increase in worm paralysis. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 100 μM Mn(III)PyP, 35 μM iron and 35 μM iron + 100 μM Mn(III)PyP. Paralysis was scored every 24 h for 5 days. Data are mean ±SEM, N=5 independent biological replicates (where one biological replicate contains 20 worms per plate). ****p<0.0001, one-way ANOVA, Tukey post hoc test. F). Mn(III)PyP worsen non-paralyzed worm swimming rates in iron toxicity environment. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 100 μM Mn(III)PyP, 35 μM iron and 35 μM iron + 100 μM Mn(III)PyP. Non-paralyzed worms were individually transferred to plate containing 100 μl of buffer. After 30 seconds of equilibration, swimming rates were collected for 15 seconds. Data are mean ±SEM N=5 independent replicates (where 4 independent worm count constitute an N). ****p<0.0001, one-way ANOVA, Tukey post hoc test. G). Schematic diagram showing specific target of EUK 134 (SOD and Catalase mimetic) in mitochondrial redox environment. H). EUK 134 protects against iron-induced toxicity induced in worm paralysis. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 100 μM EUK 134, 35 μM iron and 35 μM iron + 100 μM EUK 134. Paralysis was scored every 24 h for 5 days. Data are mean ±SEM, N=5 independent biological replicates (where one biological replicate contains 20 worms per plate). ns not significant ***p=0.0001, ****p<0.0001, one-way ANOVA, Tukey post hoc test. I). EUK 134 restored non-paralyzed worm swimming rates in iron toxicity environment. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 100 μM EUK 134, 35 μM iron and 35 μM iron + 100 μM EUK 134. Non-paralyzed worms were individually transferred to plate containing 100 μl of buffer. After 30 seconds of equilibration, swimming rates were collected for 15 seconds. Data are mean ±SEM N=5 independent replicates (where 4 independent worm count constitute an N). ns not significant, **p<0.001, ****p<0.0001, one-way ANOVA, Tukey post hoc test. J). Schematic diagram showing specific target of N-Acetyl Cysteine (NAC) in mitochondrial redox environment. K). NAC protects against iron toxicity induced increase in worm paralysis. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 2.5 mM NAC, 35 μM iron and 35 μM iron + 2.5 mM NAC. Paralysis was scored every 24 h for 5 days. Data are mean ±SEM, N=5 independent biological replicates (where one biological replicate contains 20 worms per plate). *p<0.05, ***p=0.0001, ****p<0.0001, one-way ANOVA, Tukey post hoc test. L). NAC restored non-paralyzed worm swimming rates in iron toxicity environment. Staged L4 worms were transferred to plates containing 0 μM iron, 0 μM iron + 2.5 mM NAC, 35 μM iron and 35 μM iron + 2.5 mM NAC. Non-paralyzed worms were individually transferred to plate containing 100 μl of buffer. After 30 seconds of equilibration, swimming rates were collected for 15 seconds. Data are mean ±SEM N=5 independent replicates (where 4 independent worm count constitute an N). ns not significant, ****p<0.0001, one-way ANOVA, Tukey post hoc test