FIGURE 5 from p35 is a Crucial Player in NK-cell Cytotoxicity and TGFβ-mediated NK-cell Dysfunction

TGFβ induces p35 expression in a dose- and time-dependent manner. A, NK92 cells were incubated with fluorescently-labeled Jeko-1 cells and different concentrations of TGFβ for 16 hours. Cells were stained with PI and gated on labeled cancer cells via flow cytometry to measure cancer cell death. **, P < 0.01; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, one-way ANOVA with Dunnett multiple comparisons test. B, NK92 cells were incubated with different concentrations of TGFβ in complete media for 48 hours. Protein was collected and analyzed via Western blot analysis. C, NK92 cells were incubated with 5 ng/mL TGFβ in complete media for 24 hours with or without the TGFβ receptor inhibitor EW-7197 (5 ng/mL). Protein was collected and analyzed via Western blot analysis. D, 1° NK cells were incubated in complete media with different concentrations of TGFβ and/or 5 ng/mL EW-7197 for 48 hours. Protein was collected and analyzed via Western blot analysis. E, 1° NK cells were cultured in cytokine-free media for 48 hours, followed by culture with either 200 U/mL IL2 or 5 ng/mL TGFβ. Protein lysate was collected at the indicated timepoints.