Figure 2

ATF4 is involved in the FLT3-ITD-dependent signaling required for autophagy. (a) MOLM-14 cells were treated with PBS (control) or FLT3 inhibitor (100 nM) for 30 min, 1, 2 or 16 h and were then processed for western blot analysis of ATF4. Actin was used as a loading control (n=3). (b) MV4-11 cells were treated with FLT3 inhibitor (100 nM) for 2 h, and then processed for western blot analysis of ATF4. Actin was used as a loading control (n=3). (c) MOLM-14 and MV4-11 cells were treated with PBS (control) or FLT3 inhibitor (100 nM) for 6 h then processed for Taqman qPCR analysis of ATF4. GUSB and B2M were used as control genes. Histograms represent the percentage of ATF4 mRNA in cells treated with PBS or FLT3 inhibitor (n=6). (d, e) MOLM-14 cells were treated with cycloheximide (CHX: 50 μg/ml) for 20, 40, 60, 80 or 100 min in the presence (right panel) or absence (left panel) of FLT3 inhibitor (100 nM), before being processed for western blot analysis of ATF4. Actin was used as a loading control. The graph in (e) represents the percentage of ATF4 protein remaining in cells treated with PBS or FLT3 inhibitor in presence of cycloheximide over time (n=3). (f, g). After a pretreatment with cycloheximide (50 μg/ml) of 2 h, MOLM-14 cells were washed to remove cycloheximide, and then treated with MG132 (10 μg/ml), for 1 h, 2 h or 3 h in the presence or absence of FLT3 inhibitor (100 nM), before being processed for western blot analysis of ATF4, eIF2α and P-eIF2α. Actin was used as a loading control. Histogram (g) represents the percentage of ATF4 protein in cells treated with PBS or FLT3 inhibitor in presence of MG132 over time (n=3). (h) MOLM-14 cells were transfected with ATF4-specific siRNA, then 16 h later cells were treated or not with bafilomycin (20 nM) for 2 h, before being processed for western blot analysis of ATF4 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.). (i, j) MOLM-14 cells were transfected with ATF4-specific siRNA, then 16 h later cells were treated or not with bafilomycin (20 nM) for 2 h before staining for LC3B. Representative confocal pictures are shown in (i), and the graph in (j) represents the number of LC3B dots per cell. Scale bar: 10 μm. (k) MOLM-14 cells stably expressing ATF4-specific-inducible shRNA were treated with PBS (control) or doxycycline (2 μg/ml) for 2 days. For the final 2 h cells were treated with bafilomycin (20 nM) or not and then processed for western blot analysis of ATF4 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.). PBS, phosphate buffered saline.