a. Annexin V/propidium iodide flow cytometry dot plots show marked induction of apoptosis (right two quadrants of each panel) after c-CBL knockdown. In CTCL lines (MyLa, Hut-78) which express high baseline FAS death receptor, the effect is maximal with c-CBL knockdown alone. In the other samples, treatment with MTX (which upregulates low baseline FAS death receptor expression) enhances the effect of c-CBL knockdown in CTCL lines (HH, SZ4) and SS leukemic blood samples (P1, 2). a) NS siRNA, b) MTX, c) c-CBL siRNA, d) MTX plus c-CBL siRNA. Results representative of triplicate experiments.
b. MyLa and Hut-78 CTCL lines show marked apoptosis after c-CBL siRNA knockdown regardless of whether cells are treated with anti CD3/CD28 antibodies (CD+ vs CD−) but are insensitive to antibody treatment alone. In contrast, normal CD4+ blood T-cells (N) are sensitive to antibody treatment alone as well as c-CBL knockdown. a) and c) NS siRNA, b) and d) c-CBL siRNA. Results representative of duplicate experiments.