Gene inactivations that inhibit cytoprotective responses.

Expression of stress-responsive promoter::gfp fusions was quantified following treatment with an inducing toxin or genetic disruption of insulin/IGF-1 signaling. The ER UPR gene hsp-4 is induced by the inhibitor of N-linked glycosylation tunicamycin (column 1), the Mt UPR gene hsp-6 by the ETC complex III inhibitor antimycin (column 2), the superoxide dismutase sod-3 by temperature sensitive inactivation of the daf-2(e1370) insulin/IGF-1 receptor (column 3) and the detoxification and oxidative stress response gene gst-4 by the ETC complex IV inhibitor sodium azide (column 4). Treatments were applied to day 1 adult animals raised at 20°C. RNAi clones targeting ∼1500 candidate genes were screened for suppression of GFP expression under inducing conditions. All candidates were screened in three primary replicates of 50 animals and phenotypes were verified in five or more additional replicates. Phenotypes of 29 gene inactivations found to inhibit the induction of at least one cytoprotective pathway were quantified using the automated Molecular Devices ImageXpress Micro imaging platform. Fold decrease in fusion gene expression was quantified against vector-treated controls. Data represents the fold decrease in median expression in 4 to 8 replicates of 50 animals. For clarity, results that did not meet our threshold of significance (ns) are not shown. The symbol >>> denotes that sample fluorescence values did not differ significantly from background, precluding the quantification of fold change. We identify 15 genes required for expression of hsp-4 following treatment with tunicamycin (column 1), 14 genes required for expression of hsp-6 following treatment with antimycin (column 2), 14 genes required for expression of sod-3 in a temperature shifted daf-2ts mutant (column 3), and 15 genes required for expression of gst-4 following treatment with sodium azide (column 4) with significant overlap amongst functions (Figure S3). Results include well-studied canonical regulators of cytoprotective functions, including ire-1, daf-16 and skn-1, which act specifically in the hsp-4, sod-3 and gst-4 stress response pathways, respectively. Regulation of the expression of the heat shock response phsp-16.2::gfp in response to 1 hour treatment of 37°C and expression of a constitutively expressed non-stress-responsive psur-5::gfp were quantified as controls (Table S4). Expression of endogenous loci was quantified by qPCR (Table S5).