FLT3 inhibition–induced ceramide-mediated lethal mitophagy requires Drp1 S637 dephosphorylation and activation. (A) Western blot measuring p-Drp1 S616 and p-Drp1 S637 in cells treated with FLT3 inhibitor for 6 hours. (B) Western blot detecting Drp1 in mitochondrial and cytosolic fraction after 6 hours of crenolanib treatment. Tom20 was used as a marker for mitochondria and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a marker for cytosolic fraction. (C) EM visualization of crenolanib-treated cells gold labeled with Drp1. (D) ACO2 and LC3B in crenolanib-treated sh-scr vs sh-Drp1 cells. (E) Western blot to measure CerS1 in mitochondrial and cytosolic fractions obtained from crenolanib-treated sh-scr and sh-Drp1 cells. (F) C₁₈-ceramide measured by MS HPLC-MS-MS in mitochondrial and cytosolic fraction purified from crenolanib-treated sh-scr and sh-Drp1 cells. (G) Percentage of viability measured using the MTT assay in sh-scr, sh-Drp1, sh-Drp1+Drp1WT, sh-Drp1+Drp1S637A, and sh-Drp1+Drp1S637D cells in response to 24-hour treatment with 5 μM crenolanib. (H) Western blot to detect LC3B in sh-Drp1 cells reconstituted with Drp1S637A (A) or Drp1S637D (D) mutants. (I) Western blot to detect CerS1 in purified mitochondrial fractions from sh-Drp1 cells reconstituted with Drp1 S637A (A) or Drp1 S637D (D) mutants. Tom20 and actin levels were used as positive and negative controls, respectively. Values indicate mean ± SD of n = 3 independent experiments. *P value of <.05 using the 2-sided Student t test.