(A) Cells were treated for 24 hr with 2 mM histidinol (HisOH) to mimic amino acid starvation or 300 nM thapsigargin (Tg) to induce the unfolded protein response. RNA was isolated from treated and control cells, and canonically spliced ULBP1 mRNA levels were determined by RT-qPCR. Expression levels were normalized to ACTB, GAPDH, and HPRT1 and are shown as mean ±SE. Expression in untreated WT cells was set to ‘1.0’ for each cell type; the different cell types are not comparable in this experiment. For reference, the Cq values for ULBP1 in untreated WT cells were 32.2 for K-562 cells, 27.9 for HAP1 cells, and 30.5 for Jurkat cells. The data were analyzed by 2-way ANOVA with Bonferroni's multiple comparisons test and are representative of three independent experiments, though in one of the three analyses of ATF4 KO Jurkat cells, histidinol-treated cells trended higher than untreated cells but did not reach significance. *p < 0.05, ***p < 0.001, n.s.: not significant. (B, C) Flow cytometric analysis of ULBP1 (B) and HLA Class I expression (C) on cells treated with histidinol as in (A). (D, E) Quantification of surface staining shown in (B) and (C). Data are plotted as the geometric mean fluorescence intensity of the specific stain minus the intensity of the isotype control (ΔgMFI). Data are representative of three independent experiments.