Bortezomib induces FLT3-ITD degradation through autophagy in leukemic cells. (A) Colocalization of FLT3-ITD and LC3-positive structures. MV4-11 cells were treated 12 hours with 10 nM bortezomib (Bort) before immunofluorescence analysis using LC3-B, FLT3, anti-mouse-Alexa 488, anti-rabbit A594 antibodies, and labeling with 4,6 diamidino-2-phenylindole. FLT3-ITD primary AML sample #8 was treated 12 hours with 30 nM bortezomib and analyzed by immunofluorescence with LC3-B and FLT3 antibodies. Analyses were performed on a Zeiss Apotome microscope. (B) Detection of FLT3-ITD in autophagosomes. MV4-11 cells were treated 12 hours with 10 nM bortezomib, fixed, and analyzed by electron microscopy using the Tokuyasu method. FLT3-ITD was labeled using an anti-FLT3 antibody and a secondary antibody coupled to gold beads. Observations were realized on a JEOL JEL-1400 transmission electron microscope. (C-D) Autophagy inhibition rescues FLT3-ITD protein expression. (C) MV4-11, MOLM-14, or patient sample #2 were treated 24 hours with 10 nM or 30 nM bortezomib before cell lysis and immunoblotting analysis using indicated antibodies. (D) Atg5 or Atg13 shRNA were induced by 72 hours treatment with 1 µg/mL doxycycline. Cells were then incubated 24 hours with 10 nM bortezomib and total cell lysates were analyzed by western blot.