Fig 1: Mitochondrial import of CerS1 from smooth ER mediates lethal mitophagy.(A) Confocal images of UM-SCC-22A cells induced for expression of CerS1 by Tet or SoSe and stained for CerS1 (green) and mitochondrial marker Tom20 (red) compared with nontreated cells. Yellow shows colocalization. Images represent three independent experiments. (B) Quantification of images in (A). Data are means ± SD (n = 3 different optical fields, **P < 0.01). (C) Distribution of CerS1 between mitochondria (M) and ER in UM-SCC-22A cells induced for CerS1 expression with Tet (left) or SoSe (right). Cox IV mitochondrial marker shows equal protein loading. Images are representative of three independent experiments. (D) Quantification of (C) at 72 hours for Tet-treated cells (left) and at 3 hours for SoSe-treated cells (right). Data are means ± SD (n = 3 independent experiments, **P < 0.01). (E) Quantification of confocal images of UM-SCC-22A cells treated with 5 µM SoSe for indicated periods of time and labeled with ER (PDI) and mitochondrial (Tom20) markers. Quantification of colocalization was performed with Fiji ImageJ software using at least three random fields of view from three independent experiments. Data are means ± SD (n = 3 independent experiments, **P < 0.01). (F) Quantification of the confocal images of live MEFs, from CerS1WT and CerS1Top/Top animals, treated with SoSe for indicated periods of time. MEFs were coloaded with 0.5 µM MTR for 60 min and LTR (0.5 µM for 20 min) upon treatment with 5 µM SoSe. Time points selected to illustrate onset and completion of mitochondrial digestion by autophagy. Data are means ± SD (n = 3 independent experiments, **P < 0.01). (G) Protein abundance of transiently expressed CerS1WT in MEFsTop/Top was detected by Western blotting. Actin was used as loading control. (H) Quantification of confocal microphotographs of live MEFs from animals expressing nonactive CerS1 mutant (MEFs CerS1Top/Top) and transiently transfected with empty vector control (CerS1Top/Top) or CerS1WT and treated with SoSe and stained with LTG and MTR. Time points selected to illustrate onset and completion of mitochondrial digestion by autophagy. Quantification was done using at least three images from three independent experiments. Data are means ± SD (n = 3 independent experiments, **P < 0.01). (I) Mitochondrial versus ER localization of transiently expressed FLAG-tagged CerS1WT in CerS1Top/Top MEFs after induction with SoSe. Images represent at least three independent experiments.