Skip to main content
. 2021 Dec 15;12:7310. doi: 10.1038/s41467-021-27597-7

Fig. 5. R887A mutant IRE1α displays cellular deficiency in oligomerization and RIDDLE.

Fig. 5

a Western blot analysis of endogenous and ectopic IRE1α variant expression in MDA-MB-231 cells harboring Dox-inducible IRE1α shRNA stably transfected with transgenic WT or R887A mutant versions of IRE1α-GFP. b Immunoblot analysis of MDA-MB-231 cells after treatment with Tg (100 nM, 4 h) followed by DSS crosslinking. Left panel shows parental shIRE1α cl.12 cell line. Right panel shows IRE1α WT and R887A rescues of Doxycycline-treated cl.12 cells with endogenous IRE1α knockdown. c RT-qPCR analysis of IRE1α RNase targets CD59, TGOLN2 (RIDD), and TNFAIP8L1, SNN, and SIX2 (RIDDLE). Ct values for XBP1 in sample shIRE1 cl.1 prior to Tg treatment were >34, precluding ratio calculations and were therefore not plotted. n = 3 biologically independent experiments. Data are presented as mean values ± SEM. A 2-way ANOVA test was used to calculate p-values for CD59 and TGOLN2, and an unpaired t-test for the remaining targets. d Analysis of cell viability by Cell-Titer Glo after Dox treatment for 7 days on Ultra-Low Attachment (ULA) plates. n = 2 biologically independent experiments. Data are presented as mean values ± SEM. e Model depicting IRE1α’s principal modes of endoribonuclease function and their underlying phospho-oligomeric states during ER stress. *P ≤ 0.05; **P ≤ 0.01.