Figure 2. H828Q substitution in CUA-1 increases Cu toxicity in C. elegans.

A, B. Wild-type (WT) and mutant (H828Q) animals were directly grown in CuCl₂-supplemented plates for 3 days (1^st generation, F1 protocol; see Methods). Alternatively, 3 day interval of CuCl₂ incubation was used for animals, whose parents were also grown in the presence of CuCl₂ before egg laying (2^nd generation, F2 protocol; see Methods). The plates were analyzed under the stereomicroscope (A) to quantify number of adults in the population (B). Both micrograms (A) and quantification (B) indicate that 100 μM Cu reduced number of mutant adults in the first generation, while even lower Cu concentrations significantly delayed development of mutants to adult stage in the second generation (** p<0.01, **** p<0.0001; two way ANOVA; n≥3 experiments). C. The graphs show survival curves of WT and mutant animals grown in the presence of different Cu concentrations. Both 50 and 100 μM Cu significantly shortened the lifespan of mutant animals (**** p<0.0001; Mantel-Cox test; n≥20 animals). D. Brood size was calculated for individual WT and mutant animals grown on plates containing 100 μM Cu. Quantification reveals reduction in the number of eggs deposited by mutants (**** p<0.0001; t-test; n=10 animals). E. The graph shows that daily rate of egg laying is reduced in mutant animals. F. Number of “bag worms” was calculated in the populations of WT and mutant animals grown on plates containing 100 μM Cu. The graph shows that mutant population contains more bag of worms (*** p<0.001, **** p<0.0001; two way ANOVA; n≥3 experiments). Scale bar: 1.2 mm (A)