Reduced uptake of toxic and fluorescent heme analogs may explain marginal differences observed between wildtype C. elegans and nematodes lacking HRG-9 and HRG-10 (double knockout; DKO).

A) Survival of N2, DKO, or eat-2 knockout worms exposed to 1, 2, 5, or 10 µM GaPP for 72 hours. N=3 independent experiments. * p<0.05, **** p<0.0001 B) Quantification of fluorescent staining in N2, DKO, and eat-2 worms under fed and starved conditions with or without 40 µM ZnMP treatment. A.U. arbitrary units, N=15 worms analyzed over 3 independent experiments. * p<0.05.

DKO nematodes demonstrate lawn avoidance and reduced pharyngeal pumping, brood sizes, motility, and survival.

A) Proportion of N2 or DKO worms present on OP50 lawn (innermost ring), off OP50 lawn (middle ring), or missing or dead from NGM plate (outer ring). N=180 worms over 3 independent experiments B) Number of of pharyngeal pumps in a one-minute period in N2, DKO, and eat-2 knockout worms. N=15 worms over 3 independent experiments C) Number of viable offspring laid by single adult N2 or DKO worms either after 24 hours of egg lay or across total egg-laying period (5 days). N=8 broods for 24-hour counts, N=5 broods for total brood size counts. D) Swimming behavior of N2 and DKO worms over a 20 minute interval. Worms were observed at 4 minute intervals and scored from 0-5 on swimming intensity. Bars represent the proportion of worms at each score. E) Quantification of thrashes after 4 minutes in M9 buffer. F) Longevity of N2 and DKO C. elegans observed from L4 larval stage. N=30 worms over 3 independent experiments. (** p<0.01, *** p<0.001, **** p<0.0001).

RNA-seq and qPCR analysis show that hrg-9 and hrg-10 are not uniquely heme responsive and may be linked to oxidative stress.

A) Analysis of top 500 genes with differential expression under low heme (2 µM) and high heme (400 µM) conditions. Outline represents 134 relevant genes identified by cluster analysis. B) Gene ontology analysis identified a variety of biological roles for genes within this cluster C) RT-qPCR of hrg-9 and hrg-10 under non-heme stress conditions: 24-hour starvation, 4-hour exposure to 34º C heat, and 25 mM paraquat. N=3 independent experiments.

Growth and muscle fiber integrity in yeast and zebrafish models of TANGO2 deficiency fail to replicate previously reported phenotypes.

A) Yeast growth curves. Different strains were grown in SC medium or SC medium lacking histidine at 25º C. B) Whole mount phalloidin staining of control and two strains of tango2−/−zebrafish (bwg210 and bwg211). Myofibers in mutants lack the parallel organization observed in controls but do not demonstrate significant myofiber breakdown or damage. Representative images; N=8-10 in each group. Scale bar=5 μm.