Figure 5. IRE1 supports NK cell OXPHOS and mitochondrial function.

(a) Venn diagram of RNA-seq analysis showing differentially expressed genes in IRE1^NK Ly49H⁺ NK cells compared to WT counterparts during infection with MCMV. Cells were harvested from IRE1^NK (CD45.2): WT (CD45.1) mixed BM chimeras at three time points: day 0, 1.5 and 7 PI. n = 3 mice/group (b) Top 10 enriched GSEA hallmark gene sets in RNA-seq analysis at day 1.5 PI as indicated in a. n = 3 mice/group. (c) The heat map of differentially expressed OXPHOS target genes in IRE1^NK versus WT NK cells at day 1.5 PI. Genes were clustered by functional annotation. c-Myc-regulated genes³⁵ are shown in black text on the right. (d) Analysis of NK cell oxygen consumption rate (OCR) to assess rates of OXPHOS and maximal respiration. Primary human NK cells isolated from PBMC were incubated with IL-12 and IL-18 for 16 hr, in the presence or absence of the IRE1α inhibitor 4μ8C (5 uM). Approximately 600, 000 cells were plated per well, and the data were normalized to total protein quantification. Oligo (Oligomycin, 1 μM), FCCP (carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone, 1 μM), R (rotenone, 0.5 μM) and A (antimycin, 0.5 μM). Representative plot in d shows data from one PBMC donor with each symbol representing mean measurement of three technical replicates. Quantification in d shows the combined data from three PBMC donors and two independent experiments; two-tailed unpaired Student’s t-test was performed, with error bars representing mean with s.e.m. and ****p<0.0001.