Inhibition of FLT3 induces mitophagy. (A) MV4-11 cells are pretreated with bafilomycin, inhibitor of autophagy, followed by treatment with 10 μM sorafenib, 10 μM AC220, or 5 μM crenolanib for 24 hours. Cell viability is measured using the MTT assay. (B) Western blot to detect LC3B-I (top band) and LC3B-II (bottom band) in MV4-11 cells transfected with FLT3 siRNA. (C) Western blot measuring LC3B in cells treated with crenolanib FLT3 inhibitor. (D) MV4-11 cells were transfected with FLT3 siRNA and visualized for morphology. White arrows indicate autophagosomes. (E) MV4-11 or Molm-14 cells were treated with crenolanib and gold labeled with LC3B. White arrows indicate LC3B immune-gold label in autophagosomal structures. (F) Disrupted cells were incubated with agarose beads and LC3B antibody to pull down autophagosomes followed by western blotting for Atg5, LC3B, and Tom20 in purified autophagosomes. (G) Confocal microscopy for treated cells dual labeled with LC3B antibody and Tom20 mitochondrial marker. (H) Western blotting measuring ACO2, Tom20, and PDI in crenolanib-treated cells. (I) Electron microscopy visualization of autophagosomes in crenolanib-treated cells gold labeled with Tom20 or COX-17. Squares or small black arrows indicate gold label. White arrow indicates autophagosomes. (J) Western blot analysis for LC3B in sh-scr and sh-LC3B stable-transfected cells. (K) Percentage of viability measured using the MTT assay in sh-LC3B cells treated with different doses of crenolanib for 24 hours. (L) Percentage of nonviable cells measured using MTT assay in sh-scr, sh-LC3B, sh-LC3B+WTLC3B, and sh-LC3B+LC3BG120A. (M-N) Western blot to measure ACO2 and LC3B-II in crenolanib-treated sh-scr vs sh-CerS1 cells. *P value of <.05 using the 2-sided Student t test. All images are representative of at least 2 independent experiments.