Molecular and Cellular Biochemistry

, Volume 465, Issue 1–2, pp 53–64 | Cite as

Characterization of IRE1α in Neuro2a cells by pharmacological and CRISPR/Cas9 approaches

  • Kentaro Oh-hashiEmail author
  • Hiroki Kohno
  • Mahmoud Kandeel
  • Yoko Hirata


IRE1 is the most conserved endoplasmic reticulum (ER)-resident stress sensor. Its activation not only splices XBP1 but also participates in a variety of cell signaling. We elucidated the role of IRE1α in Neuro2a cells by establishing IRE1α-deficient cells and applying four IRE1 inhibitors. IRE1α deficiency prevented almost all spliced XBP1 (sXBP1) protein expression by treatment with thapsigargin (Tg) and tunicamycin (Tm); these phenomena paralleled the values measured by our two Nanoluciferase-based IRE1 assays. However, cell viability and protein expression of other ER stress-responsive factors in the IRE1α-deficient cells were comparable to those in the parental wild-type cells with or without Tm treatment. Next, we elucidated the IRE1 inhibitory actions and cytotoxicity of four compounds: STF083010, KIRA6, 4μ8C, and toyocamycin. KIRA6 attenuated IRE1 activity in a dose-dependent manner, but it showed severe cytotoxicity even in the IRE1α-deficient cells at a low concentration. The IRE1α-deficient cells were slightly resistant to KIRA6 at 0.1 μM in both the presence and absence of ER stress; however, resistance was not observed at 0.02 μM. Treatment with only KIRA6 at 0.1 μM for 12 h remarkably induced LC3 II, an autophagic marker, in both parental and IRE1α-deficient cells. Co-treatment with KIRA6 and Tm induced LC3 II, cleaved caspase-9, and cleaved caspase-3; however, IRE1α-deficiency did not abolish the expression of these two cleaved caspases. On the other hand, KIRA6 prohibited Tm-induced ATF4 induction in an IRE1-independent manner; however, co-treatment with KIRA6 and Tm also induced LC3 II and two cleaved caspases in the ATF4-deficient Neuro2a cells. Thus, we demonstrate that IRE1α deficiency has little impact on cell viability and expression of ER stress-responsive factors in Neuro2a cells, and the pharmacological actions of KIRA6 include IRE1-independent ways.


ER stress IRE1 XBP1 



Activating transcription factor 4


Activating transcription factor 6


Endoplasmic reticulum


Growth arrest and DNA damage inducible gene 153


78 kDa glucose-regulated protein


94 kDa glucose-regulated protein


Glyceraldehyde 3-phosphate dehydrogenase


Inositol-requiring enzyme-1


PKR-like endoplasmic reticulum kinase


Regulated IRE1-dependent mRNA decay


X-box binding protein 1



This work is, in part, is supported by Grant-in-aid from the Japan Society for the Promotion of Science (JSPS, Japan, KAKENHI, Nos. 17K19901 and 19H04030 to K.O.). We are grateful to Dr. George Church for providing the hCas9 gene.

Author contributions

KO and MK discussed and designed the research; KO and HK performed experiments; KO and YH wrote the manuscript.

Compliance with ethical standards

Conflict of interest

There was no conflict of interest in this study.

Supplementary material

11010_2019_3666_MOESM1_ESM.pptx (287 kb)
Supplementary material 1 (PPTX 287 kb)
11010_2019_3666_MOESM2_ESM.docx (82 kb)
Supplementary material 2 (DOCX 83 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kentaro Oh-hashi
    • 1
    • 2
    • 3
    Email author
  • Hiroki Kohno
    • 2
  • Mahmoud Kandeel
    • 4
    • 5
  • Yoko Hirata
    • 1
    • 2
    • 3
  1. 1.United Graduate School of Drug Discovery and Medical Information SciencesGifu UniversityGifuJapan
  2. 2.Graduate School of Natural Science and TechnologyGifu UniversityGifuJapan
  3. 3.Department of Chemistry and Biomolecular Science, Faculty of EngineeringGifu UniversityGifuJapan
  4. 4.Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary MedicineKing Faisal UniversityHofufSaudi Arabia
  5. 5.Department of Pharmacology, Faculty of Veterinary MedicineKafrelsheikh UniversityKafrelsheikhEgypt

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