Figure 5

Inhibition of upstream BRCA1 phosphorylation effectors ATR/ATM, decreases Tat-dependent transcription. A. TZM-bl cells were transfected with pcTat and treated the next day with vehicle (water) and a titration of caffeine (500 μM, 2 mM and 5 mM). Bright-Glo luciferase assays were performed 48 hours post-treatment. Data was normalized to cells containing Tat and treated with DMSO as baseline for Tat-dependent LTR activation. B. CellTiter-Glo cell viability assays were performed 48 hours post-treatment. Data was normalized as in panel A. C. TZM-bl cells were transfected with pcTat and treated the next day with vehicle (DMSO) and a titration of ATM or Chk2 inhibitors (ATMin and Chk2in) at 0.1 μM, 1 μM and 10 μM. Bright-Glo luciferase assays were performed 48 hours post-treatment. Data was normalized as in panel A. D. CellTiter-Glo cell viability assays were performed 48 hours post-treatment. Data was normalized as in panel A. E. TZM-bl cells were co-transfected with pcTat or siRNA against GFP (control) and ATM. Bright-Glo luciferase assays were performed 48 hours post-treatment. Cells containing pcTat and siGFP were used as baseline value for Tat-dependent LTR activation. ATM depletion was confirmed by qRT-PCR and western blot (right panel and inset). Fold changes against siGFP were calculated relative to Actin using the ΔΔCt method. F. TZM-bl cells were transfected with pcTat and treated the next day with DMSO or ATM inhibitor (10 μM) for 48 hours prior to being collected for ChIP analysis. Antibodies used for ChIP were anti-BRCA1 (10 μg), anti-V5 (10 μg), and anti- pS10-H3 (10 μg). Transfection and treatment assays were performed in triplicate and data represents averaged data of two independent experiments. Viability assays were performed in triplicate. Error bars show the standard error of two averaged independent measurements. Double asterisk indicates statistically significant difference p ≤ 0.01.