Fig. 1: FLT3 inhibitors induce autophagy in FLT3-ITD models.

A Dose-response curve for midostaurin and quizartinib treatment in MOLM13 and MV4-11 cells. Analysis was performed by methylthiazoletetrazolium (MTT) assay for MOLM13 cells (blue) and MV4-11 cells (red) after 48 h of treatment with vehicle, midostaurin (3.125, 6.25, 12.5, 25, 50, and 100 nM) or quizartinib (0.625, 1.25, 2.5, 5, 10, and 20 nM). Values are expressed as the percentage of viable cells for each condition relative to untreated controls. Data are presented as the mean of at least three independent experiments. In MOLM13 cells, the median inhibitory concentration (IC₅₀) of midostaurin was 25.5 nM, whereas in MV4-11 cells, the IC₅₀ was 41.8 nM. The IC₅₀ of quizartinib was 1.8 nM in MOLM13 cells and 2.327 nM in MV4-11 cells. B Treatment with FLT3i increased the frequency of acidic vesicular organelles (AVO) detected by flow cytometry in MOLM13 and MV4-11 cells treated with vehicle or graded concentrations of midostaurin (6.25, 12.5, and 25 nM) or quizartinib (0.62, 1.25, and 2.5 nM) for 48 h using the acridine orange staining method. Representative dot plots are shown for each condition; the “AVO” gate contains cells positive for the FL-3 channel, which is characterized by increased acidic vesicular organelle formation and acridine orange color shift. Bar graphs represent the mean and standard deviation of at least three independent experiments; p values are indicated on the graphs. *p < 0.05; **p < 0.01, for cells treated compared with vehicle; ANOVA test and Bonferroni post-test. C Treatment with FLT3i increased the formation of autophagosomes in MOLM13 and MV4-11 cells, visualized by the color change of acridine orange (green to orange) upon pH changes during the formation of acidic autophagic organelles. Slides were prepared after 48 h of treatment with vehicle, midostaurin (12.5 nM) or quizartinib (1.25 nM), and after 15 min of incubation with the acridine orange probe. D Analysis of the dose-dependent effect of midostaurin and quizartinib on the protein expression of MOLM13 and MV4-11 cells. Western blotting analysis was performed on total cell extracts of cells treated with vehicle, midostaurin (6.25, 12.5, and 25 nM) or quizartinib (0.62, 1.25, and 2.5 nM) for 48 h. E Analysis of the effect of midostaurin and quizartinib over time on protein expression of MOLM13 and MV4-11 cells. Western blotting analysis was performed on total cell extracts of cells treated with vehicle, midostaurin (12.5 nM) or quizartinib (1.25 nM) for 0, 3, 6, 9, 12, 24, and 48 h.