Oncogenic KIT mutations induce STAT3-dependent autophagy to support cell proliferation in acute myeloid leukemia

a–d Oncogenic KITD816V drives autophagy. a TF-1 or TF-1 KITD816V cells were incubated for 4？h with PBS, Bafilomycin A1 (20？nM, n？=？3？±？SEM), or chloroquine (20？μM, n？=？2？±？SD), and were then analyzed by immunoblotting using the appropriate antibodies. Numbers represent the LC3B-II/actin ratios obtained by densitometric analysis. b Cells were incubated for 4 or 8？h with vehicle or 20？μM of chloroquine, and then incubated with Cyto-ID before flow cytometry (n？=？3？±？SEM). ΔMFI？=？MFI(chloroquine)？？？MFI(vehicle). c Cells were treated with vehicle or BafA1 for 2？h before LC3 staining, fluorescent labeling, and immunofluorescence analysis. d Quantification of LC3-positive autophagosomes (n？=？3？±？SEM). e, f Pharmacological inhibition or genetic invalidation of a decrease in KIT autophagic flux. e TF-1 KITD816V cells were treated overnight with PKC412 at 1？μM; 20？nM of BafA1 was added at 2？h before cell lysis and western blotting. Numbers represent the LC3B-II/actin ratios obtained by densitometric analysis (n？=？2？±？SD). f KITD816V cells were incubated for 3 days with doxycycline to induce expression of shRNA against KIT. Cells were treated for 2？h with BafA1 (20？nM) and then analyzed by western blotting. Numbers represent the LC3B-II/actin ratios obtained by densitometric analysis (n？=？2？±？SD