Inhibition of TRAIL-induced apoptosis requires ABL kinase activity and nuclear localization.

<div><p>a. Schematic representation of the Type VI mAbl cDNA constructs used in reconstitution experiments. WT, wild type; KD, kinase defective (K291H); µNLS, nuclear localization signals mutated (11 K/R substitutions with Q); µNES, nuclear export signal mutated (L1064A). </p> <p>b. Levels of endogenous ABL and mAbl proteins in whole cell lysates of the indicated HT29 cells. </p> <p>c. Immunofluorescence staining of the indicated HT29 cells with anti-ABL (green) and DAPI (blue). Scale bar - 20 µm.</p> <p>d. ABL knockdown enhanced, and reconstitution with mAbl, suppressed TRAIL-induced sub-G1. Cells treated with the indicated doses of TRAIL were collected at 16 hr and analyzed for sub-G1 by FACS. Values shown are mean and standard deviation from five independent experiments. **** = p < 0.00005 and ** = p < 0.005. </p> <p>e. ABL knockdown reduced survival. Cells were treated with 100 ng/ml Tr daily for three days, replated in media without Tr for another 3-days and cell survival measured by crystal violet assay (see Materials and Methods). Values shown are mean and standard deviation from three independent experiments each with four repeats. * p = 0.03. </p> <p>f. Inhibition of TRAIL-induced apoptosis requires mAbl kinase activity and nuclear import. Cells were treated with the indicated doses of Tr as in (d). Values shown are mean and standard deviations from 5 independent experiments. ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005.</p>