Journal: Molecular Oncology
Article Title: Synergistic induction of apoptosis by the Bcl‐2 inhibitor ABT‐737 and imatinib mesylate in gastrointestinal stromal tumor cells), Synergistic induction of apoptosis by the Bcl‐2 inhibitor ABT‐737 and imatinib mesylate in gastrointestinal stromal tumor cells
Figure Lengend Snippet: Combined treatment with ABT‐737 and imatinib induces apoptosis synergistically to overcome imatinib‐resistance in GIST48IM cells. (A) The antiproliferative effect of single‐agent imatinib (left) and single‐agent ABT‐737 (right) in imatinib‐resistant GIST48IM cells was examined after 24, 48 and 72 h of treatment, using the MTS cell viability assay. Columns, mean of duplicate experiments; error bars, SD. Results were analyzed by two‐way ANOVA. (B) The effect of combined ABT‐737 (0, 0.1, 1, 10, 20 μM) and imatinib (0, 0.1, 1, 10 μM) on the viability of GIST48IM cells at 72 h. Columns, mean of duplicate experiments; error bars, SD. Results were analyzed by one‐way ANOVA. (C) Normalized isobologram (top) and Fa‐CI plot (bottom) of GIST48IM cells, graphically depicting synergistic, additive, and antagonistic interactions between imatinib and ABT‐737 in this cell line. (D) To determine whether reductions of GIST48IM cell viability were due to apoptosis, nuclear morphology was assessed by EB/AO staining after treatment with ABT‐737 and imatinib for 72 h. Representative micrographs of ethidium bromide/acridine orange‐stained GIST48IM cells. Original magnification, ×200. Abbreviations: (N), normal nuclei; Thick arrow, late apoptosis; (A), apoptotic nuclei; Thin Arrow, early apoptosis. (E) Quantification of normal and apoptotic cells treated with 1 μM imatinib alone, or combined with ABT‐737 (0.1, 1, 10, 20 μM). (F) Immunoblot analysis of the expression of Bcl‐2, Bcl‐xL and Mcl‐1, as well as the cleavage of caspase 3 and PARP, after treatment with DMSO, 1 μM imatinib, 10 μM ABT‐737, or a combination for 72 h. Actin was used to demonstrate equal loading. Abbreviations: (F), full length; (C), cleaved.
Article Snippet: Primary antibodies used to detect poly‐ADP‐Ribose polymerase (PARP) (rabbit polyclonal, #9542; 1:1000), caspase 3 (mouse monoclonal, #9668; 1:1000), Bcl‐2 (rabbit monoclonal, #2870; 1:1000), Bcl‐xL (rabbit monoclonal, #2764; 1:1000), and Mcl‐1 (rabbit polyclonal, #4572; 1:1000) were procured from Cell Signaling Technology (Danvers, MA).
Techniques: Viability Assay, Staining, Expressing