Dose- and time-dependent expression of IFN-γ and STAT4 in IL-12-treated NK cells. (A) NK cells were treated with 0, 0.1, 1, or 10 ng/mL IL-12 and incubated for the times indicated in the presence of IL-2. Culture supernatants were collected at the times indicated points from 2 to 32 hours. The IFN-γ levels in the medium were quantified by ELISA. Bars at 2, 4, and 8 hours indicate cumulative IFN-γ levels in culture supernatants; Bars at 16, 24 and 32 hours indicate IFN-γ production in each preceding 8 hours in culture supernatants. (B) NK cells were treated with 0 or 10 ng/mL IL-12 and incubated for indicated times in presence of IL-2. Total RNA was purified from the respective cell pellets and analyzed by qRT-PCR for the expression of IFN-γ mRNA. (C) NK cells were cultured as in panel A. Cells were collected at the times indicated and whole-cell extracts were prepared. Western blots were performed with anti-STAT4. (D) NK cells were treated with 0 or 10 ng/mL IL-12 and incubated for indicated times in presence of IL-2. Total RNA was purified from the respective cell pellets and analyzed by qRT-PCR for the expression of STAT4 mRNA. “2 + 12 / 2” represents the fold change in mRNA levels calculated by comparing the value of IL-2 + IL-12–cultured cells (2 + 12) to that of IL-2-cultured samples²  in parallel. The results are mean ± SEM of representative experiments with NK cells from 3 different donors. *P < .05, **P < .01 vs 16 hours (A) or 8 hours (B) same concentration of IL-12 treated cells. ^#P < .05, ^##P < .01 versus 0 hours untreated cells.