Fig 2: IRE1 phosphorylates SPL during ER stress. A: HEK293 cell were cotransfected with IRE1 and FLAG-SPL plasmids and treated with TG (600 nM; 4 h). IRE1 and SPL were immunoprecipitated with total IRE1 and FLAG antibodies, respectively, and analyzed by Western blotting using antibodies specific for total IRE1 and FLAG. Normal IgG Rabbit or normal IgG-Mouse antibodies were used to show specifity of IRE1 and FLAG antibodies, respectively. The cell lysate was also analyzed by Western blotting using antibodies specific for p-IRE1, total IRE1, and FLAG (n = 4 biological replicates, a representative blot is shown). B: In vitro kinase assay: HEK293 cells were transfected with the indicated plasmids and treated with TG (1 μM; 2 h). IRE1 and SPL were immunoprecipitated with FLAG antibodies and incubated together with ATPγS in a kinase reaction. The kinase reaction was analyzed by Western blotting using specific antibodies for thio-phospho ester and FLAG (n = 3 biological replicates, a representative blot is shown). C: “Cell-based” kinase assay: HEK293 cells were transfected with the indicated plasmids and treated with TG (1 μM; 2 h) followed by treatment with N6 furfuryl-ATPγS. Immunoprecipitated lysates were analyzed by Western blotting with specific antibodies for thio-phospho ester, total IRE1, and FLAG (n = 3 biological replicates; a representative blot is shown). D: Multiple amino acid sequence alignment of SPL from different species: ∗ indicates possible IRE1-mediated phosphorylation sites as determined by mass spectrometry. E: In vitro kinase assay: HEK293 cells were transfected with the indicated plasmids and treated with TG (1 μM; 2 h). IRE1 and SPL were immunoprecipitated with total IRE1 and FLAG antibodies, respectively, and incubated together with ATPγS in a kinase reaction. The kinase reaction was analyzed by Western blotting using specific antibodies for thio-phospho ester, total IRE1 and FLAG (n = 3; a representative blot is shown). ER, endoplasmic reticulum; HEK293, human embryonic kidney 293; IRE1, inositol-requiring enzyme-1; SPL, sphingosine 1-phosphate lyase; TG, thapsigargin.

Fig 3: IRE1-SPL axis impinges on eif2α phosphorylation through modulating PERK and PKR activities and establishes an additional layer of control over UPRmt induction during ER stress in mammalian cells. Activated by ER stress, IRE1 phosphorylates SPL that is found on the ER membranes. IRE1-mediated phosphorylation leads to inactivation to SPL and increased S1P levels in cells. IRE1-SPL axis mainly regulates PKR activity and eif2α phosphorylation, a critical upstream event in the activation of mammalian UPRmt signaling. ER, endoplasmic reticulum; IRE1, inositol-requiring enzyme-1; mt, mitochondria; PKR, double-stranded RNA-activated protein kinase; PERK, PKR–like ER kinase; SPL, sphingosine 1-phosphate lyase; UPR, unfolded protein response.

Fig 4: Measuring recovery of IRE1 clusters by FRAP. (A) Representative image of an IRE1-mNG cell where 1 large and 2 small clusters are bleached with a focused 405-nm laser beam. Images at the top are zoomed-in views of the recovering cluster, indicated by the red squares in the images below. Inset in the 0-s panel shows the same cluster with lower brightness correction to illustrate that the cluster is nearly diffusion-limited in size and only appears large in the other images due to saturation. All images are γ-corrected with a γ-value of 1.3 to make the faint outline of the ER easier to see. (Scale bars: Top, 2 μm; Bottom, 10 μm.) (B) Schematic depiction of the local background subtraction strategy for monitoring FRAP in recovering clusters. First, the recovering cluster is tracked throughout the movie, yielding a diffusion trajectory (positions and trajectory are colored by frame time). Then, cluster intensity (inner circles, bottom row) is measured and corrected for local background (blue outer circles, bottom row). (C) Representative FRAP trace (blue line) and fit (red line) of a bleached IRE1 cluster. Inset shows an expanded view of the recovery, which plateaus at ∼3% of the initial intensity and is difficult to see on the full-scale graph. (D) Representative FRAP trace of unclustered IRE1 from a 5-μm radius bleached region in a cluster-free region of the ER. (E) Comparison of recovery half-time measurements of clustered and unclustered IRE1. (F) Comparison of mobile fraction extracted from FRAP curves of clustered and unclustered IRE1.

Fig 5: siRNA mediated knockdown of JNK, IRE1 blunted HCV up-regulation of NFκB activation.Huh7.5.1 cells and Huh7.5.1 cells infected with JFH1 for 3 days were transfected with 25 nM small interference RNA (siRNA) against JNK, IRE1, ATF6, and PERK. After 48 hours, the expression of phospho-JNK, phospho-IRE1, phospho-PERK, ATF6 cleavage and phospho-NFκB were analyzed by western blot. JNK siRNA decreased JFH1-induced IRE1, PERK, NFκB protein phosphorylation and ATF6 activation. IRE1 knockdown decreased HCV-induced NFκB phosphorylation in JFH1-infected Huh7.5.1 cells (Fig. 9A). We added 20 μg of protein to each well to assure equal protein loading in each lane. Lane 1, Huh7.5.1; lane 2, JFH1 + NTsiRNA; lane 3, JFH1 + JNKsiRNA; lane 4, JFH1 + IRE1siRNA; lane 5, JFH1 + ATF6siRNA; lane 6, JFH1 + PERKsiRNA. We found that siRNA knockdown of the tested genes did not affect cell viability compared to negative control siRNA (Fig. 9B).