Expression of STAT4 in cells treated in vitro with chemotherapy drugs. PBMCs obtained from controls were activated with PHA and IL-2 for 3 days and then cultured for 3 more days with the indicated concentrations of etoposide (Eto) and carmustine (Car). RNA was extracted and the first-strand cDNA was synthesized from the cells. STAT4 expression was analyzed using immunoblot (A) and real-time PCR (B). (C) STAT4 protein levels in CD4⁺ and CD8⁺ cells from control activated PBMCs treated without (-D) or with carmustine (Car) or etoposide (Eto) were analyzed using flow cytometry as described in “Methods.” Histograms represent the STAT4 expression gated on 5000 events of live CD4⁺ or CD8⁺ cells using WinMDI software. (D) NKL cells were treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) for 2-3 days. STAT4 protein levels were analyzed using Western blotting. (E) NKL cells treated as described in panel D were incubated with medium alone (unfilled bars) or medium containing 2 ng/mL of IL-12 (filled bars) for 1 day. The cell-free supernatants were analyzed for IFN-γ production using ELISA. The data are presented as means ± SD from 3 independent experiments.