Figure 1

FLT3-ITD supports basal autophagy in leukemic cells. (a) MOLM-14 cells were treated with PBS (control) or FLT3 inhibitor (100 nM) for 16 h in the presence or absence of chloroquine (15 μM), before being processed for western blot analysis of phospho-STAT5 (p-STAT5), STAT5 and LC3B. Actin was used as a loading control. Numbers represent the LC3B-II/actin ratios obtained by densitometric analysis (n=3±s.e.m.). (b, c) MOLM-14 cells were treated with PBS (control) or FLT3 inhibitor (100 nM) for 16 h in the presence or absence of chloroquine (15 μM), then stained for LC3B. Representative confocal pictures are shown in (b), and the graph in (c) represents the number of LC3B dots per cell. Scale bar: 10 μm. (d) MV4-11 cells stably expressing FLT3-specific-inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 2 days, then bafilomycin (20 nM) was added or not for 2 h before the cells were processed for western blot analysis of FLT3 and LC3B. Actin was used as a loading control. Numbers represent the LC3B-II/actin ratios obtained by densitometric analysis (n=3±s.e.m.). (e, f) MV4-11 cells stably expressing FLT3-specific-inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 2 days, then chloroquine (15 μM) was added for 16 h before staining for LC3B. Representative confocal pictures are shown in (e), and the graph in (f) represents the number of LC3B dots per cell. Scale bar: 10 μm. (g) Patient samples harboring the FLT3-ITD mutation were treated with PBS (control) or FLT3 inhibitor (100 nM) for 16 h in the presence or absence of chloroquine (10 μM), before being processed for western blot analysis of STAT5, phospho-STAT5 (p-STAT5) and LC3B. Actin was used as a loading control. Numbers represent the LC3B-II/actin ratios obtained by densitometric analysis (n=1). (h) Same experiment as in (g), performed with primary cells from two patients expressing wild-type FLT3. PBS, phosphate buffered saline.