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

Figure 1. Anti‐apoptotic role of MITOL against ER stress.

Figure 1

  1. MITOL‐KO MEFs were vulnerable to ER stress. Control MEFs (MITOLF/F) and MITOL‐KO MEFs (MITOL−/−) were treated with DMSO as control, 0.8 μM thapsigargin (Tg), 0.7 μg/ml tunicamycin (Tu), or 1.2 μg/ml brefeldin A (Br) for 24 h. All experiments using Tu below were performed at concentration of 0.7 μg/ml. The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers. Viable cells were detected by cell viability assay using Cell Counting Kit‐8. Error bars represent SD (n = 6). n is the number of independent experiments. ***P < 0.001 (Student's t‐test).
  2. MITOL KO increased ER stress‐induced apoptosis. MEFs were treated with indicated ER stress inducers at same concentration as Fig 1A for 18 h and stained with Annexin V‐FITC for the detection of apoptosis using flow cytometry. Error bars represent SD (n = 3). *P < 0.05 (Student's t‐test).
  3. MITOL re‐expression in MITOL‐KO MEFs rescued ER stress vulnerability. MEFs were transfected with MITOL‐coding vector (WT), MITOL C65/68S‐coding vector (CS), or empty vector (Vec) 24 h prior to Tu treatment for 18 h, followed by immunoblotting with indicated antibodies. Cleaved caspase‐3 (cC3) and cleaved PARP (cPARP) were used as apoptosis markers. Error bars represent ± SD (n = 3). **P < 0.01, ***P < 0.001 (Student's t‐test).
  4. MITOL KO enhanced ER stress‐induced Bax translocation and cytochrome c release. Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 h, followed by immunoblotting with indicated antibodies. Error bars represent SD (n = 3). **P < 0.01, ***P < 0.001 (Student's t‐test).
  5. MITOL KO induced excessive mitochondrial depolarization by ER stress. MEFs transfected with indicated vectors were treated with Tu for 18 h and then stained with TMRM, followed by flow cytometric analysis. Error bars represent SD (n = 3). *P < 0.05 (Student's t‐test).