Figure 6

ABL1 is localized with F-actin structures in CLL cells and the substrate molecule CRKL undergoes increased phosphorylation. Panel A. Dual-colour Immunocytofluorescence of CLL lymphocytes probed for F-actin (red) and ABL1 (green)(representative images of CLL lymphocytes with morphological features typical of amoeboid motility). F-actin is particularly enriched within extended projections at the anterior pole of the cell (left panel). ABL1 protein (right panel) shows a fraction with typical nuclear localisation, but an significant additional cytoplasmic fraction (right panel) is co-localised with F-actin (co-incident pixels are shown as yellow in central combined image). This is further highlighted by pixel colocalisation studies, for the image shown, co-localised pixels are indicated in white (ImageJ/FIJI software Colocalisation plugin). Panel B. Phosphorylated CRKL protein is increased within cultured cells (mean fluorescence [MF] indicated with each panel). (i) CLL B-cells isolated directly from circulation express low levels of 207P-CRKL. (ii) Cells from culture show an increased 207P-CRKL expression (solid histogram) compared with cells from blood (open histogram) (iii) The large irregular cells (high side scatter fraction) from culture have highest 207P-CRKL expression. Panel C. 207P-CRKL phosphorylation does not vary significantly between cases of CLL with IgVH mutation (M-CLL) or without IgVH mutation (UM-CLL); alpha tubulin is shown as a loading control. Panel D. Imatinib mesylate inhibits 207P-CRKL-phosphorylation in a dose dependent manner (samples treated with CXCL12 or imatinib at indicated concentration for 30 minutes, 207P-CRKL immunoblot imatinib doses as indicated, alpha tubulin is shown as a loading control).