STAT1 Regulates Human Glutaminase 1 Promoter Activity through Multiple Binding Sites in HIV-1 Infected Macrophages (original) (raw)
Figure 3
STAT1 binds directly with several binding sites of the GLS1 promoter in
IFN-α treated and HIV-1 infected cells. (A) THP1 cells were treated with 100 U/ml IFN-α for indicated times, then p-STAT1 (Tyr 701), and STAT1 were detected by Western blot. β-actin was used as a loading control. (B) A schematic diagram of the primers used in ChIP assay covering sites 2/3 and 5/6. (C, D) STAT1 binds with sites 2/3 and 5/6 in human GLS1 promoter in THP1 cells. THP1 cells were treated with 100 U/ml IFN-α for one hour, then ChIP assay was performed using digested chromatin, STAT1 antibody, and IgG antibody as a negative control. Purified DNA was analyzed by real-time RT-PCR using specific primers for sites 2/3 (C) and sites 5/6 (D). The amount of immunoprecipitated DNA was normalized as a ratio to the total amount of input chromatin and shown as fold change relative to control without treatment. The data are representative of three independent experiments. (E, F) STAT1 binds with sites 2/3 and 5/6 in human GLS1 promoter in MDM cells. MDM were treated with 100 U/ml IFN-α for one hour or infected with HIV-1ADA for five days. ChIP assay was performed using STAT1 antibody as described in (C). The data are representative of three independent experiments using three different donors. *, p < 0.05, **, p < 0.01, ***, p < 0.001 when compared with the control without IFN-α treatment.