Figure 3

IL-15 priming induced HIF-1α accumulation and glycolysis in NK cells depend on STAT3. (A–I) NK cells were incubated under normoxia (N) or hypoxia (H) for 16 h and primed with IL-15 for additional 6 h in the presence or absence of STAT3 inhibitors (S3I-201, stattic) or rapamycin (rapa) as indicated. (A) Bead array analysis of pSTAT3^Ser727. Mean values ± SD of median fluorescence intensities (MFI) are shown (n = 4). The level of statistical significance was not reached when applying the Wilcoxon signed-rank test. (B) and (C) Flow cytometric data for the proportions of (B) pSTAT3^Ser727 MFI and (C) the proportion of pSTAT3⁺ NK cells, shown as mean values ± SD (n = 3). *p < 0.05, Friedman test with Dunn’s test for post-hoc pairwise comparisons. (D) HIF1A gene expression analysis by RT-PCR. Linearized quantities (2^−ΔCt) are represented as mean values ± SD (n = 9). **p < 0.01, Wilcoxon signed-rank test. (E) Immunoblot analysis with antibodies against HIF-1α and β-actin. The blot reflects one representative experiment of three performed. (F) and (G) Glycolytic parameters. Extracellular acidification rate (ECAR) values were measured over time in a hypoxic chamber at 1% O₂. Each data point represents an average ± SD of three independent experiments. Dashed lines indicate additions of glucose (Gluc), oligomycin (Oligo), and 2-deoxyglucose (2-DG). *p < 0.05, Friedman test with Dunn’s test for post-hoc pairwise comparisons. (H) and (I) Flow cytometric data for (H) the proportion of GLUT1⁺ hypoxic NK cells and (I) GLUT1 MFI is represented as mean values ± SD (n = 4). The level of statistical significance was not reached when applying the Wilcoxon signed-rank test.