STAT1 phosphorylation is required for IFN-α to activate the GLS1 promoter and induce glutaminase expression and function.

<p>(A). MDM were treated with different doses of IFN-α for 24 hours, then p-STAT1 (Tyr 701), STAT1, KGA and GAC were detected by Western blot. β-actin was used as a loading control. (B). Intracellular glutamate was detected using the Amplex^® Red Glutamic Acid/Glutamate Oxidase Assay Kit. The data are representative of three independent experiments using three different donors and are the means of triplicate samples. (C-F). Levels of p-STAT1 (C), STAT1 (D), GAC (E) and KGA (F) in Western blot (A) were normalized as a ratio to β-actin and shown as fold change relative to control. Results are shown as the average ± SEM of three independent experiments with three different donors, (G, H). Correlation of p-STAT1 with GAC (G) and p-STAT1 with KGA (H) in representative donor are shown. (I, J). HEK 293T cells were co-transfected with the GLS1 promoter construct and pRL-SV40. 24 hours later, the cells were pretreated with 1 µM fludarabine or 1:10,000 DMSO for 1 hour, then treated with or without 100 U/ml IFN-α for 24 hours. (I). p-STAT1 and STAT1 were detected by Western blot, β-actin was used as a loading control. (J). Luciferase activity in the lysates was measured by luminescence detection. Renilla luciferase was used to normalize transfection efficiency. The data are representative of three independent experiments and are the means of triplicate samples. #, p<0.05. *, p<0.01, **, p<0.001 when compared to control.