Figure 3.

C₁₈-ceramide accumulates in mitochondria and binds to LC3B to recruit autophagosomes to mitochondria. (A) MV4-11 or Molm-14 cells are fractionated to purify mitochondrial and cytosolic fractions followed by western blot to detect CerS1 in the purified fractions. Tom20 was used as a marker for mitochondrial fraction (M) and actin was used as a marker for cytosolic fraction (C); (B) HPLC-MS-MS measurement of C₁₈-ceramide in mitochondrial and cytosolic fractions. (C) Electron microscopy (EM) visualization of MV4-11 cells treated with crenolanib and gold labeled with CerS1 antibody. Black arrows indicate gold label in mitochondria and white arrows indicate autophagosome. (D) EM visualization of MV4-11 or Molm-14 cells treated with crenolanib and gold labeled with ceramide antibody. Black arrows indicate gold label in mitochondria and white arrows indicate autophagosomes. (E) Treated sh-scr and sh-CerS1 cells are dual labeled with ceramide antibody and Tom20 mitochondrial marker and visualized using confocal microscopy. White arrows indicate colocalization. The quantification of ceramide-Tom20 colocalization from confocal microscopy was performed using the ImageJ Fiji software (right panel). (F) Crenolanib-treated MV4-11 cells are dual labeled with ceramide antibody and LC3B autophagosomal marker and visualized using confocal microscopy. White arrows indicate colocalization. (G) Western blot to detect LC3B protein in sh-LC3B cells reconstituted with LC3B WT or LC3B mutants (I35A and F52A) that cannot bind ceramide. (H) Percentage of viability measured using MTT assay for sh-scr, sh-LC3B, sh-LC3B+LC3B WT, sh-LC3B+LC3B I35A, and sh-LC3B+LC3BF52A, treated with crenolanib for 24 hours. (I) Autophagosomes were purified from vehicle or crenolanib treated in sh-LC3B cells reconstituted with LC3B WT or LC3B mutants (I35A and F52A) that cannot bind ceramide. This was followed by a western blot to detect autophagosome markers (Atg5 or LC3B) and Tom20 mitochondria marker. Values indicate mean ± SD of n = 3 independent experiments. *P value of <.05 using the 2-sided Student t test. C, cytosolic fraction; M, mitochondrial fraction; W, whole cell.