Figure 3

BRCA1 status affects enhancement of Tat-dependent transcription during infection with pseudotyped particles. A. Schematic depicting the LTR-driven reporter plasmid pNL-RRE-SA-Luc. B. UWB1.289 BRCA1 null and UWB1.289 + BRCA1 cells and were co-transfected with pcTat and CMV-Luc (Renilla) plasmid DNA 24 hours prior to infection with LTR-driven reporter viral particles. Dual-Glo luciferase assay was performed 24 hours post-infection as described by the manufacturer. Raw data was normalized to Renilla luciferase expression in both cell lines, fold changes were calculated against + Tat mock infected cells (lane 1). Transfection and infection assays were performed in triplicate and data plotted represents averaged data of two independent experiments. Single asterisk indicates p < 0.05. C. UWB1.289 BRCA1 null and UWB1.289 + BRCA1 cells were infected with VSVG-pseudotyped NL4-3. Cells were collected 48 hours post-infection, stained with propidium iodine, and analyzed by flow cytometry. Results are representative of three biological replicates. D. Cells were infected as described above and collected at 48 hours post-infection for ChIP analysis. Antibodies used for ChIP were anti-RNA polymerase II (RNAP II, 10 μg), anti-Sp1 (10 μg), and anti-V5 (10 μg). Quantitative PCR was performed using SYBR Green PCR Master Mix to analyze immunoprecipitated material.