Journal: Molecular and Cellular Biology
Article Title: The N-Terminal Domain of p73 Interacts with the CH1 Domain of p300/CREB Binding Protein and Mediates Transcriptional Activation and Apoptosis
Figure Lengend Snippet: p300 enhances p73-induced apoptosis. (A and B) The N-terminal deletion of p73 lacks apoptosis-promoting potential. As indicated, plasmids encoding no protein as a control (C; 3 μg), p73α (1.0 μg), ΔNp73α (1.0 μg), and/or p300 (2.0 μg), together with the pGFP plasmid (200 ng), were introduced into H1299 cells (10 5 /60-mm-diameter dish) using LipofectAmine (GIBCO-BRL); 32 h posttransfection, cells (100) expressing GFP were counted under a fluorescence microscope using a blind approach, with morphologically round cells (arrows) identified as apoptotic (A). (B) Statistical data (with standard error indicated by a bar above each column) from three independent assays. (C and D) Caspase 3 activation by wild-type but not N-terminus-deleted p73α is enhanced by p300. Transient transfection as described for panel A was conducted. Cells were harvested for caspase 3 activity analysis as described in Materials and Methods. (C) Representative result of the fluorescence graph of AMC released from Ac-DEVD-AMC substrates for one set of experiments with plasmids encoding no protein (curve B), ΔNp73α (curve C), ΔNp73α plus p300 (curve D), p73α (curve E), or p73α plus p300 (curve F). Curve A is a water control. Caspase 3 activity is presented in arbitrary units (A.U.) after normalization by a factor of 10 5 . (D) Statistical values for caspase 3 activity in triplicate assays. (E) DNA fragmentation analysis of p73-induced apoptosis in H1299 cells was conducted as described in Materials and Methods; 10 μg of DNA was analyzed by electrophoresis on 2% agarose. Each lane represents the result for three 60-mm-diameter plates of cells. MW, molecular weight markers.
Article Snippet: H1299 cells (50% confluent) were transfected with plasmids as indicated in the legend to Fig. , using LipofectAmine (Promega) as described above.
Techniques: Plasmid Preparation, Expressing, Fluorescence, Microscopy, Activation Assay, Transfection, Activity Assay, Electrophoresis, Molecular Weight