Hlx inhibits IFN-γ mRNA and protein expression in NK-92 cells. (A) Overexpression of Hlx protein in NK-92 cells. FACS-purified NK-92 cells transduced with PINCO or PINCO-Hlx were subjected to nucleocytoplasmic fractionation and immunoblotting for Hlx. Enrichment of nuclear protein was confirmed by Brg1 staining. Equal loading was confirmed by Ku70 staining. (B) Inhibition of IFN-γ production by Hlx. PINCO- or PINCO-Hlx–infected NK-92 cells were stimulated with IL-12/IL-18, and supernatants were harvested after 24 hours for IFN-γ ELISA. Shown are the mean ± SEM from 5 separate experiments. (C) Time course of IFN-γ mRNA in PINCO-compared with PINCO-Hlx–transduced NK-92 in response to IL-12/IL-18. Shown are results of 1 of 4 representative experiments; error bars represent SD of triplicate PCR reactions. (D) Increased IFN-γ production by Hlx^−/− NK in response to monokine costimulation. Fetal liver–derived NK of indicated genotypes were subjected to 24-hour stimulation with IL-12 and IL-15, followed by intracellular staining for IFN-γ. Mean ± SEM percentages of IFN-γ⁺ NK1.1⁺ cells from 9 experiments with NK cells derived from 9 or more livers of each genotype are shown. *P < .001 Hlx^+/+ compared with Hlx^−/−. **P < .005 Hlx^+/− compared with Hlx^−/−).