C22:0- and C24:0-dihydroceramides Confer Mixed Cytotoxicity in T-Cell Acute Lymphoblastic Leukemia Cell Lines
Figure 5
Caspase cleavage and LC3B-I/II turnover.
A) CCRF-CEM cells treated with drug/fatty acid vehicle (C), sphinganine (1 µM, S), or sphinganine (1 µM) + GT-11 (0.5 µM) (SG), were supplemented with the indicated fatty acids (5 µM). After 12 hours, total proteins were extracted and procaspase-3 (35 kb), activated caspase-3 (17/19 kb), and LCB3-I/II (14/16 kb), were detected by immunoblotting. β-Actin served as loading control. Treatment with pan-Bcl-2 inhibitor, ABT-737 (1 µM, A), and LC3B-transfected, HEK-293 cell lysate, were used as positive controls. C22:1-fatty acid was used as a negative control for C22:0-fatty acid. Lanes rearranged to ease interpretation. Data representative of three separate experiments are shown. B) Assessment of LC3B-II flux. CCRF-CEM cells were pretreated with or without protease inhibitors (Pepstatin-A and E64d) and treated as described. After 12 hours, total proteins were extracted and LC3B-I/II analyzed by immunoblotting. Data representative of three separate experiments are shown, except for C22:1-fatty acid.