To compare the efficacy of using different target genes to monitor activation of the mitoUPR, we examined the expression of each gene under conditions of ATFS-1 activation. To activate ATFS-1, we used a nuo-6 mutation (blue bars) or the constitutively active atfs-1(et15) and atfs-1(et17) mutations (green bars). ATFS-1 activation was prevented using an atfs-1 deletion mutation gk3094 in wild-type and nuo-6 worms (red bars and purple bars, respectively). Expression is shown as a percentage of wild type worms (white bars). A. There is a significant increase in hsp-6 mRNA under conditions of ATFS-1 activation that is prevented by disruption of atfs-1.
B. The levels of hsp-60 are not increased in nuo-6 mutants or either constitutively active atfs-1 mutants. C-F.The mitoUPR target genes C01G1.7, F22B3.7, cyp-14A4 and clec-17 exhibit a marked increased in expression under conditions of ATFS-1 activation with a magnitude much greater than hsp-6.
G-J. The mitoUPR target genes hrg-9, K09E9.1, cdr-2 and F15B9.10 show a large increase in expression relative to their standard deviation under conditions of ATFS-1 activation. K-N. The expression of mitoUPR target genes cdr-2, F15B9.10, C07G1.7 and hrg-9 show increased expression in nuo-6 worms by quantitative RT-PCR. O. Heat map comparing the expression of identified mitoUPR target genes between wild-type and long-lived mitochondrial mutant isp-1 worms. All but 4 of the 61 genes are significantly upregulated in isp-1 mutants. Expression levels in panels A-J were determined from our previously published RNA sequencing data (Wu et al., 2018). Expression represents counts per million from six biological replicates that has been normalized to wild-type. Heat map was generated using our previously published RNA sequencing data (Senchuk et al., 2018). Error bars indicates SEM. **p<0.01, ***p<0.001. Statistical significance was assessed using a one-way ANOVA with Dunnett’s multiple comparison test, except in panels K-N where a student’s t-test was used.