l-ase induces strong protective autophagy in AML cells. (A-B) OCI-AML3 cells were cultured for 6 or 24 hours in 10% FCS MEM with or without 0.1, 1, and 10% FCS MEM with or without 0.1, 1, or 10 UI/mL of E colil-ase and with or without Gln as indicated. Autophagy was detected by western blotting using anti-LC3b and p62/SQSTM1 antibodies. (C) Representative electron micrographs of OCI-AML3 cells collected after 24 hours of culture in MEM with or without E colil-ase (1 UI/mL) and without glutamine. Arrows indicate autophagic vesicles. Upper and lower panels show 2 different scales. (D) OCI-AML3 cells were transfected with anti-ATG5 or anti-beclin siRNAs using the Amaxa nucleofector kit. Two days after transfection, cells were cultured for 24 hours with or without E colil-ase at 10 UI/mL and western blotting was performed to verify ATG5 and Beclin knockdowns, respectively. (E) Viability was measured in OCI-AML3 cells (transfected or not with anti-ATG5 or anti-Beclin siRNA and treated or not with E colil-ase) by flow cytometry analysis of Annexin V fixation. The histograms shown represent the mean values of 3 independent experiments.