FIG 5.

DNAPK activity is not required for GR-mediated BRG1-dependent transcriptional activation from chromatinized MMTV or endogenous promoters. (A) SW-13/MMTV cells expressing GR and BRG1 were treated with 25 μM DNAPK-specific inhibitor NU7026 (+NU7026) or DMSO vehicle (−NU7026) for 24 h. After exposure, cells were treated with Dex or vehicle (EtOH) for 6 h, followed by total RNA extraction and cDNA synthesis. Equal amounts of cDNA and gene-specific primers for luciferase, HSD11B2, or PLZF were used for real-time PCR analysis. Quantitative analysis was performed with data normalized to GAPDH and displayed as relative expression. Values are shown as mean ± standard deviation (n = 3). (B) To evaluate the effectiveness of NU7026, cells were pretreated with NU7026 or vehicle (DMSO), followed by irradiation with 5 Gy IR. At 15 min post-IR, cells were fixed, permeabilized, and stained with anti-gamma-H2A.X (phosphor S139) antibody. Representative images, collected using an LSM 5 Pa confocal microscope at ×63 magnification, are shown for each treatment group. Scale bars, 10 μm.