Stable knockdown of ABL enhances TRAIL-induced apoptosis.

<div><p>a. Doxorubicin and TRAIL caused ABL down-regulation. HT29 cells pretreated with doxorubicin (Dox, 2.0 µg/ml, 24 hr) or not were treated with TRAIL (Tr - 200 ng/ml, 8 hr) or not and whole cell lysates were immunoblotted with the indicated antibodies. </p> <p>b. ABL knockdown stimulated TRAIL-induced caspase activation. The indicated HT29 cells were treated with 200 ng/ml TRAIL. Whole cell lysates were collected at the indicated time points and blotted with the indicated antibodies. </p> <p>c. ABL levels in HT29 cells without (-) or with (+) the ABL-shRNA or the wild type (WT) mouse type-IV Abl (mAbl). The indicated amounts of whole cell lysates (µg) were loaded in each lane. </p> <p>d. Re-expression of mAbl in ABL-knockdown cells suppressed TRAIL-induced caspase activation. Caspase and PARP1 cleavage in the indicated HT29 cells treated with doxorubicin (2 µg/ml) and/or TRAIL (200 ng/ml) for 8 hrs.</p> <p>e.,f. DEVDase and IETDase activity in the indicated HT29 cells treated as in (d) with doxorubicin and/or TRAIL. ABL-knockdown significantly enhanced TRAIL-induced DEVDase (e) and IETDase (f). Reconstitution of mAbl expression significantly reduced DEVDase (e) and IETDase (f). Values shown are mean and standard deviation from three independent experiments each with duplicate measurements. * = p < 0.05; ** = p <0.01. </p> <p>g. Annexin V staining of the indicated HT29 cells treated as in (d) with doxorubicin and/or TRAIL. ABL-kockdown significantly enhanced TRAIL-induced Annexin V positivity. Reconstitution of mAbl expression significantly reduced Annexin V positivity. Values shown are mean and standard deviations from three independent experiments each with duplicate measurements. * = p < 0.05. </p>