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. 2013 Oct 18;8(10):e76941. doi: 10.1371/journal.pone.0076941

Figure 5. Mitochondria-derived oxidative factor activates IRE1.

Figure 5

(a) Effects of Tg, antimycin A (AMA), or rotenone on the activation of ER stress sensors. IRE1 were immunoprecipitated before detection by western blottings. No active form of ATF6 (p50) or phosphorylated PERK was detected under the experimental condition with AMA or rotenone. (b) Effects of NAC on phosphorylation of IRE1 caused by AMA. (c) Effects of AMA on the splicing of the XBP1 mRNA in CHO cells. Un, unspliced; S, spliced. (d) AMA (1 µM) induced the expression of XBP1-venus in an IRE1-dependent manner. CHO cells were transfected with FLAG-XBP1-venus plasmids and siRNA (siCon, scrambled siRNA; siIRE1, IRE1 siRNA) two days before the AMA treatment. Induction of FLAG-XBP1-venus proteins was measured by a fluorescence microplate reader. ***P<0.001 (n = 12), compared with siCon. (e) Selective activation of IRE1 by AMA in the MAM-containing crude mitochondrial fraction. AMA-treated CHO cells were homogenized and subjected to differential centrifugation. The asterisk indicates the hyperphosphorylated form of IRE1. (f) Effect of mifotusin-2 knockdown on the AMA-induced IRE1 phosphorylation. siRNA against mitofusin-2 (siMFN2) or scrambled control siRNA (siCon) was transfected into CHO cells two days before the treatment with AMA. In the graph, IRE1 was normalized to ERK and shown as means±S.E.M. **p<0.01 (n = 4) compared with siCon without AMA. (g) AMA induces a reduction of pIRE1 proteins in CHO cells with reduced expression of Sig-1Rs. Control or Sig-1R siRNA were transfected into CHO cells two days before AMA. IRE1 was immunoprecipitated before detection.