S1 File - Genkwadaphnin Induces IFN-γ via PKD1/NF-κB/STAT1 Dependent Pathway in NK-92 Cells

Cytotoxicity, optimum culture conditions and concentrations of GD-1 on IFN-γ production in NK-92 cells. S1 Figure, Determination of effective concentration of GD-1 for IFN-γ production in human myeloid cell lines and comparison between GD-1 and other stimuli, CD40, LPS, TNFα on IFN-γ release from NK-92 cell. A. Evaluation of IFN-γ production in the human myeloid cell lines, U937, HL-60, THP-1, K562, and NK-92. All ELISA data are representative of at least three independent experiments. B. Evaluation of IFN-γ production in the NK-92 cell line in presence of immune stimuli, CD40 (1∶10 ng/ml, 2∶1 ng/ml), LPS (1:100 ng/ml, 2∶10 ng/ml), TNFα (1∶5 ng/ml, 2∶0.5 ng/ml) and GD-1 (1∶2µg/ml, 2∶200 ng/ml). All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. *P<0.05. S2 Figure, Cytotoxicity of GD-1 during IFN-γ production under GD-1 treated NK-92 cell. A. Cell proliferation was measured using the WST-1 reagent (Roche Applied Science) according to the manufacturer's protocol. NK-92 cells were seeded about 2x104 cells per well in 100µl media volume on a 96 well flat-bottom plate. After seeding cells, wells were treated with the indicated concentration of GD-1 for 12hr. GD-1 have no significant cytotoxicity on the NK-92 in the dose range from 0 to 200 ng/ml. B. NK-92 cell was treated GD-1 under condition of serial concentrations from 0 to 200 ng/ml for 12hr. IFN-γ production by GD-1(100 ng/ml) in culture supernatant was saturated. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. *P<0.05. S3 Figure, Evaluation of secreted cytokines production in the GD-1 treated human myeloid cells. The secreted IL-12 (A), IL-2 (B), TNFα (C) and IL-10 (D) were examined in the GD-1 treated NK-92 cells. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. *P<0.05. S4 Figure, Optimum culture conditions for IFN-γ production under GD-1 treated NK-92 cells. GD-1-treated NK-92 cells were cultured under condition of a serial concentration of FCS (from 20% to 0%) and with (+) or without (-) of IL-2 for 12hr. All ELISA data are representative of at least three independent experiments. Triplicate samples in each time were tested and averaged. Error bars indicate standard deviation. *P<0.05.
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