PPAR-γ expression in freshly isolated human NK cells. (A) RT-PCR detection of PPAR-γ mRNA and T-bet. Freshly isolated human NK cells were treated with IL-2, IL-4, IL-12, and IL-2 plus IL-12 for 24 hours. RT-PCR was performed to detect PPAR-γ, T-bet, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Densitometry analysis was performed to quantitate the expression level of PPAR-γ as indicated. (B) Identification of PPAR-γ protein by Western blotting. Immunodetection of PPAR-γ in cell lysates of freshly isolated human NK cells, NK92 cells, NKL cells, THP-1 cells, and U937 cells. NKL cells were treated with IL-4 for 24 hours. Both THP-1 and U937 cells were treated with phorbol-12-myristate-13-acetate (PMA) for 24 hours. (C) Detection of PPAR-γ and T-bet by flow cytometry in human NK cells. CD56⁺ cells were gated on human NK cell population, and expression of PPAR-γ and T-bet was detected by flow cytometry through intracellular staining. Shaded histograms are the Ab isotype control; open histograms are anti-T-bet and anti-PPAR-γ, respectively. Brackets represent the percentage of positive cells relative to the control. Numbers above the peaks refer to the mean fluorescence intensity (MFI). (D) Relative expression levels of PPAR-γ and T-bet in NK cells from 7 donors. MFI; control IgG for T-bet: IC (T-bet) (▪); anti-T-bet: T-bet (▴); control IgG for PPAR-γ: IC (PPAR-γ) (▾); and anti-PPAR-γ: PPAR-γ (♦) are represented by in each group. Horizontal bars represent the mean of each set.