Figure 3

Targeting autophagy inhibits FLT3-ITD AML cells proliferation. (a) MOLM-14 cells were treated with DMSO (control) or the VPS34 inhibitor SAR-405 (3 μM) for 3 days. For the final 2 h cells were treated or not with bafilomycin (20 nM) and were then processed for western blot analysis of 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) MOLM-14 cells were treated with DMSO (control) or SAR-405 (3 μM) for 3 days, before the number of cells was assessed by trypan blue exclusion counting. The graph represents the number of cells per day (n=3±s.e.m.). (c) MOLM-14 cells were treated with DMSO (control) or SAR-405 (3 μM) for 3 days before cell death was assessed by annexin V labeling and flow cytometry analysis. The graph represents the percentage of annexin V-positive cells (n=3±s.e.m.). (d) MOLM-14 cells stably expressing an ATG12-specific inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 3 days. For the final 2 h cells were also treated with bafilomycin (20 nM) or not before being processed for western blot analysis of ATG12 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) MOLM-14 cells stably expressing ATG12-specific inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 3 days before the number of cells was assessed by trypan blue exclusion counting. The graph represents the number of cells per day (n=3±s.e.m.). (f) MOLM-14 cells stably expressing ATG12-specific inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 3 days before cell death was assessed by annexin V labeling. The graph represents the percentage of dead cells (n=3±s.e.m.). (g) MOLM-14 cells stably expressing ATF4-specific inducible shRNA were treated with DMSO or doxycycline (2 μg/ml) for 3 days before the number of cells was assessed by trypan blue exclusion counting. The graph represents the number of cells per day (n=3±s.e.m.). (h) MOLM-14 cells stably expressing ATF4-specific inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 3 days before cell death was assessed by annexin V labeling. The graph represents the percentage of dead cells (n=3±s.e.m.). PBS, phosphate buffered saline.