Figure 3.

Mycolactone targets primarily the IFN-γ signaling pathway in Jurkat T cells. (A) Production of IFN-γ by Jurkat T cells treated with 20 nM mycolactone (Myco) or vehicle (Ctrl) for 6 h (Resting) or for 1 h before 6 h of activation with PMA/IO. (B) Quantitation of IFNG mRNAs in Jurkat T cells treated with mycolactone or vehicle for 6 h or for 1 h before 3 or 6 h of activation with PMA/IO. (C) Western blot analysis of tyrosine phosphorylated (STAT1-P) and total STAT1 in Jurkat T cells treated with mycolactone or vehicle for the indicated times before activation with 1 ng/ml IFN-γ for 20 min or left unstimulated (Unst). (D) Flow cytometric analysis of surface expression of IFNGR1, IFNAR1, and IFNAR2 by Jurkat T cells incubated with or without mycolactone for 6 h. (E) Quantification of IFNGR1 mRNAs in Jurkat T cells treated as in B. (F and G) Quantitation of GBP2 mRNAs (F) and total GBP2 protein (G) in Jurkat T cells treated as in B. (H) Western blot analysis of phosphorylated (STAT1-P, STAT2-P, and STAT3-P) and total STAT1, STAT2, and STAT3 with β-actin as the loading control in Jurkat T cells treated with mycolactone or vehicle for 24 h before activation with 1 ng/ml IFN-γ or IFN-α for 20 min or left unstimulated (Unst). (A and D) Data are mean IFN-γ levels or mean fluorescence intensity, respectively, ± SEM of one experiment performed in triplicate, relative to vehicle controls. (B, E, and F) Data are mean fold-changes ± SEM of one experiment performed in duplicate, compared with resting controls. Similar results were obtained in independent experiments. (A, C, G, and H) Data are from one of two independent experiments, which gave similar results.