Figure 6. BRCA1 expression counteracts mutant p53 GOF activity on DNA repair assay.

(A) Comparison of ligation products of 5′-cohesive-ended linear DNA in the presence of T4 DNA ligase alone (lane 3) or following pre-incubation with whole protein extracts derived from H1299 cells transfected with mutp53R175H and BRCA1 expressing vectors in separate reactions (lanes 5 and 7, respectively) or in co-trasfection conditions (lane 6). (B) Whole protein extracts (40 μg) used in the T4 DNA ligase assay previously described were subjected to Western blot analysis and probed with the indicated antibodies. (C-E) SKBr3 cells were transiently transfected with ApaI-linearized pSI-CHECK2 vector (c) and with either siRNA oligos indicated in the figures (d) and (e). After 48 h from the transfection the cells were harvested and the functional changes in NHEJ were assessed measuring the Firefly Luciferase activity. Luciferase activity was expressed as (Firefly/protein amount) × (1/Renilla). Columns, means from two independent assays each of them was done in triplicate; bars, SD. P-values were calculated with two tailed t-test. Statistically significant results were with p-value < 0.05.