Skip to main content
. 2019 Jun 13;38(15):e100999. doi: 10.15252/embj.2018100999

Figure EV4. Defect of IRE1α ubiquitylation by MITOL leads to abnormal ER morphology, related to Fig 5 .

Figure EV4

  • A
    K481R mutation of IRE1α did not impair its RNase activity. IRE1α‐KO MEFs were transfected with indicated vectors 24 h prior to Tu treatment for 4 h. Expression levels of xbp1s mRNA were determined by qRT–PCR. Error bars represent SD (n = 3). *P < 0.05, n.s.: not significant (Student's t‐test).
  • B
    Overexpression of IRE1α K481R did not affect PERK and ATF6 pathway. MEFs were transfected with indicated vectors 24 h prior to immunoblotting with indicated antibodies. MEFs treated with Tu for 8 h were prepared as a positive control for UPR activation. cATF6: cleaved ATF6.
  • C, D
    The K481R mutation of IRE1α also induced cell death under ER stress. MEFs were transfected with indicated vectors 24 h prior to analysis. Viable cells were evaluated as similar to Fig 1A (C). Dead cells were quantified by cell toxicity assay using cytotoxicity LDH assay Kit‐WST. Vec: empty vector. Error bars represent SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t‐test).
  • E
    Overexpression of IRE1α K481R‐induced morphological change in the ER. MEFs were transfected with indicated vectors and mC‐Sec61β 24 h prior to analysis. The lower panels show fivefold magnification image of the boxed regions. Arrowheads indicate abnormal parts of the ER network. Scale bar represents 10 μm. Error bars represent SD (n = 3). *P < 0.05 (Student's t‐test).
  • F
    Overexpression of IRE1α K481R increased resting Ca2 in the ER. MEFs were transfected with G‐CEPIA1er 24 h before analysis. Resting Ca2+ in the ER was calculated from 10 cells in each independent experiment. Error bars represent SD (n = 3). *P < 0.05 (Student's t‐test).