Figure 5

Targeting autophagy or ATF4 overcomes resistance to FLT3 inhibitors. (a) MOLM-14 TKD cells were treated with DMSO (control) or SAR405 (a VPS34 inhibitor, 3 μM), for 3 days. For the final 2 h cells were then treated with bafilomycin (20 nM) or not and 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 TKD cells were treated with DMSO (control) or SAR405 (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 TKD cells stably expressing ATG12-specific inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 3 days. For the final 2 h cells were then treated with bafilomycin (20 nM) or not and 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.). (d) MOLM-14 TKD 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.). (e) NSG mice (n=15) engrafted with MOLM-14 TKD cells stably expressing the ATG12 inducible shRNAs by i.v. injection, were treated with sucrose (10 μg/ml) with or without doxycycline (200 μg/ml) via their drinking water and their overall survival was analyzed. The graph represents the Kaplan–Meier survival curves. PBS, phosphate buffered saline.