Loss of STAT3 in NK cells is accompanied by increased expression of perforin and granzyme B. (A-B) One representative western blot (n = 2) of (A) ex vivo MACS-purified and CD3⁻NKp46⁺ sorted Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg NK cells and (B) MACS-purified and IL-2–expanded Stat3^fl/fl and Stat3^Δ/ΔMx1-Cre NK cells without any stimulus or after stimulation for 30 minutes with IL-2 + IL-12, 3 hours with IL-2 + IL-12, or 30 minutes with IL-2 + IL-15. STAT3 activation, STAT3 deletion in Stat3^Δ/ΔNcr1-iCreTg NK cells, and granzyme B expression were detected. (C) MACS-purified and FACS-sorted (CD3⁻NK1.1⁺) Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg NK cells were stimulated ex vivo with IL-2 alone or together with IL-12. Gzmb and Prf1 mRNA expressions were determined by qPCR. Data represent mean ± SEM of 2 independent experiments (n ≥ 5); RNA levels are calculated relative to that of the HKG Rplp0. (D-E) Splenocytes from Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were analyzed directly ex vivo or after stimulation for 8 hours with IL-2, IL-12, and IL-15 and stained for CD3, NKp46 (CD3⁻NKp46⁺), and perforin or granzyme B. Levels were analyzed by flow cytometry. Bar graphs depict mean fluorescence intensities (MFI) (n ≥ 7 per genotype). (F) MACS-purified and FACS-sorted (CD3⁻NK1.1⁺) NK cells were treated with DMSO (control), with 0.5, 5, 10, or 20 µM of the STAT3 inhibitor LLL-12 or with 100 µM of the STAT3 inhibitor S31-201 for 4 hours. NK cells were either left untreated (IL-2 media only) or stimulated with IL-12 for 20 minutes. Western blot analysis shows levels of pSTAT3-Y705 and STAT3. β-actin was used as loading control. (G) MACS-purified and FACS-sorted (CD3⁻NK1.1⁺) NK cells were treated with DMSO, with 0.5 µM of the inhibitor LLL-12, or with 100 µM of the inhibitor S31-201 for 4 hours after stimulation with IL-2, IL-2 + IL-12, or IL-2 + IL-12 + IL-15 for 3 hours. Perforin and granzyme B mRNA levels were analyzed by qPCR. (Data represent mean ± SEM; levels are calculated relative to the HKG Rplp0.)