Figure 1. Distinct IRE-1-dependent responses to ROS and ER stress.

(A–B) IRE-1 is required for the C. elegans p38/SKN-1 response to ROS. ire-1 RNAi diminished the p38 (A) and gst-4P::GFP (B) responses to sublethal exposure to AS (30 min) or PQ (30 min), but not Sorbitol (ST, 30 min) or heat (30 min). Phosphorylation of p38 (P-p38) indicates p38 activation (Inoue et al., 2005). (C–E) Acute AS treatment does not activate the UPR^ER. AS (30 min) did not upregulate hsp-4P::GFP (C), with intestinal GFP scoring in (D), or promote IRE-1 and HSP-3/4 dissociation as determined by co-immunoprecipitation (co-IP) (E). (F–I) AS pre-treatment blocks IRE-1 and UPR^ER activation in response to ER stress. Prior AS exposure (30 min) inhibited IRE-1 phosphorylation (F), xbp-1 mRNA splicing, as indicated by quantitiative PCR assay of levels of the indicated mRNA forms (G), XBP-1S protein accumulation (H), and hsp-4P::GFP induction (I–J) by ER stress (TM; 5 hr). AS-treated samples were allowed to recover for 30 min. prior to TM exposure. (K) AS does not inhibit the PERK kinase PEK-1, as indicated by phosphorylation of its substrate eIF2-α. In the right panels PEK-1 expression was blocked by RNAi. (L–O) Pre-exposure to ER stress inhibits the antioxidant response. Exposure to TM (5 hr) prior to AS treatment (30 min.) decreased p38 activation (L), SKN-1 nuclear localization (M) and intestinal gst-4P::GFP expression (N–O). In all figures, mock incubations and zero time points are indicated by “C”. (B, D, J, M, O) GFP quantification with high (H), medium (M) or low (L) scoring. p < 0.0001****; p < 0.001***; p < 0.01**. All immunoblots in the paper are representative of at least two and in most cases three experiments. See also Figure S1.