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.

中文翻译：

---

通过药理学和CRISPR / Cas9方法表征Neuro2a细胞中IRE1α。

IRE1是最保守的内质网（ER）驻留应力传感器。它的激活不仅剪接XBP1，而且参与各种细胞信号转导。我们通过建立IRE1α缺陷细胞并应用四种IRE1抑制剂阐明了IRE1α在Neuro2a细胞中的作用。IRE1α缺乏症通过毒胡萝卜素（Tg）和衣霉素（Tm）的治疗阻止了几乎所有剪接的XBP1（sXBP1）蛋白表达；这些现象与我们两个基于纳米荧光素酶的IRE1分析所测得的值相近。但是，IRE1α缺陷型细胞中其他ER应激反应因子的细胞活力和蛋白质表达与经过或未经过Tm处理的亲本野生型细胞相当。接下来，我们阐明了IRE1的抑制作用和四种化合物的细胞毒性：STF083010，KIRA6、4μ8C和Toyocamycin。KIRA6以剂量依赖的方式减弱IRE1的活性，但即使在低浓度的IRE1α缺陷细胞中，它也显示出严重的细胞毒性。在存在和不存在内质网应激的情况下，IRE1α缺陷型细胞在0.1μM时对KIRA6均具有轻微的抗性。但是，在0.02μM下未观察到电阻。仅用0.1μM的KIRA6处理12小时，即可在亲本和IRE1α缺陷型细胞中明显诱导自噬标记物LC3 II。与KIRA6和Tm共同处理诱导LC3 II，裂解的caspase-9和裂解的caspase-3；然而，IRE1α缺乏并没有消除这两个裂解的胱天蛋白酶的表达。另一方面，KIRA6以与IRE1无关的方式禁止Tm诱导的ATF4诱导。但是，与KIRA6和Tm的共同处理也会在ATF4缺失的Neuro2a细胞中诱导LC3 II和两个裂解的半胱天冬酶。因此，