( A ) The levels of two major ceramide ( Cer ) species in lipid extracts from WT ( black ) , tul1Δ ( dark gray ) or Orm2 - K25 , 33R mutants ( light grey ) were measured using LC - MS and quantified using spiked non - yeast ceramides as internal standard . Data are normalized to Cer44 : 0 ; 4 levels of WT cells ( set to 1 ) and presented as mean ± standard deviation from three independent experiments . Pairwise statistical significance was assessed by Student ' s t - test . p values are given above the respective bars .

O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-ORGANISM B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The levels of two major ceramide (Cer) species in lipid extracts from WT (black), tul1Δ (dark gray) or Orm2-K25,33R mutants (light grey) were measured using LC-MS and quantified using spiked non-yeast ceramides as internal standard. Data are normalized to Cer44:0;4 levels of WT cells (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.


( B ) Autoradiogram of sphingolipid extracts from [ 3H ] serine radiolabelled WT cells and tul1 ∆ mutants were separated by thin layer chromatography . IPC , Inositolphosphorylceramide ; MIPC , mannosylinositolphosphorylceramide ; MIP2C , mannosyldiinositolphosphorylceramide . X indicates an unknown lipid species that is insensitive to myriocin treatment .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O

(B) Autoradiogram of sphingolipid extracts from [3H]serine radiolabelled WT cells and tul1∆ mutants were separated by thin layer chromatography. IPC, Inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; MIP2C, mannosyldiinositolphosphorylceramide. X indicates an unknown lipid species that is insensitive to myriocin treatment.


( C ) SDS - PAGE and Western blot analysis with the indicated antibodies of input and elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from cells expressing FLAG - Orm2 - WT or FLAG - Orm2 - K25 , 33R . Control cells expressed untagged Orm2 WT . The asterisks label unspecific cross - reactions of the respective antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O

(C) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-WT or FLAG-Orm2-K25,33R. Control cells expressed untagged Orm2 WT. The asterisks label unspecific cross-reactions of the respective antibodies.


SDS - PAGE or Phos - tag - PAGE and Western blot analysis with the indicated antibodies of total cell lysates ( D ) from WT cells expressing FLAG - Orm2 or FLAG - Orm2 - K25 , 33R The asterisks label unspecific cross - reactions of the respective antibodies .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (D) from WT cells expressing FLAG-Orm2 or FLAG-Orm2-K25,33R The asterisks label unspecific cross-reactions of the respective antibodies.


SDS - PAGE or Phos - tag - PAGE and Western blot analysis with the indicated antibodies of total cell lysates ( E ) from WT cells and tul1 ∆ mutants expressing FLAG - Orm2 , FLAG - Orm2 - 3A or FLAG - Orm2 - 3D with or without Myriocin treatment ( 1 . 5 µM ) for one hour .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (E) from WT cells and tul1∆ mutants expressing FLAG-Orm2, FLAG-Orm2-3A or FLAG-Orm2-3D with or without Myriocin treatment (1.5 µM) for one hour.


( A ) Left panel : In tor1 - 1 avo3 ∆ 1274 - 1430 cells rapamycin specifically inhibits TORC2 but not TORC1 ( Gaubitz et al . , 2015 ) . tor1 - 1 avo3 ∆ 1274 - 1430 cells were treated with cycloheximide ( CHX ) and rapamycin or vehicle ( methanol ) for the indicated times . Right panel : WT or Orm2 mutant cells were treated with CHX for the indicated times . The protein levels of FLAG - Orm2 or of the indicated FLAG - Orm2 mutants from total cell lysates were analyzed by SDS - PAGE and Western blotting with the indicated antibodies .

O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(A) Left panel: In tor1-1 avo3∆1274-1430 cells rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al., 2015). tor1-1 avo3∆1274-1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG-Orm2 or of the indicated FLAG-Orm2 mutants from total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.


( B ) Graphs display the FLAG - Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0 , 90 and 180 min after the addition of CHX . Each experiment was repeated at least three times . FLAG - Orm2 levels were normalized to Pgk1 loading controls , and each timepoint was related to t = 0 min ( set to 1 ) . Data are presented as mean ± standard deviation .

O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each timepoint was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.


( C ) Denaturing immunoprecipitation ( IP ) of FLAG - Orm2 , FLAG - Orm2 - 3A and FLAG - Orm2 - 3D . The control strain expresses untagged Orm2 . Input and elutions ( with FLAG peptides ) were analyzed by SDS - PAGE and Western blot with the indicated antibodies .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(C) Denaturing immunoprecipitation (IP) of FLAG-Orm2, FLAG-Orm2-3A and FLAG-Orm2-3D. The control strain expresses untagged Orm2. Input and elutions (with FLAG peptides) were analyzed by SDS-PAGE and Western blot with the indicated antibodies.


( D ) Epifluorescence and phase contrast microscopy of living tul1 ∆ mutants expressing GFP - Orm2 , GFP - Orm2 - 3A or GFP - Orm2 - 3D ( green ) with dsRed - HDEL ( red ) . Arrowheads point to GFP - Orm2 accumulations outside the endoplasmic reticulum ( ER ) . Scale bars = 5 µm .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O

(D) Epifluorescence and phase contrast microscopy of living tul1∆ mutants expressing GFP-Orm2, GFP-Orm2-3A or GFP-Orm2-3D (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Scale bars = 5 µm.


( E ) SDS - PAGE and Western blot analysis with the indicated antibodies of input and elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from cells expressing FLAG - Orm2 - K25 , 33R , FLAG - Orm2 - K25 , 33R - 3A or FLAG - Orm2 - K25 , 33R - 3D . The control strain expresses untagged Orm2 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O

(E) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-K25,33R, FLAG-Orm2-K25,33R-3A or FLAG-Orm2-K25,33R-3D. The control strain expresses untagged Orm2.


( F ) The levels of the long chain bases ( LCB ) C18 - dihydrosphingosine ( C18 - DHS ) and C18 - phytosphingosine ( C18 - PHS ) and of two major ceramide ( Cer ) species in lipid extracts from WT , tul1 ∆ , Orm2 - K25 , 33R , Orm2 - 3A and Orm2 - 3D mutants were measured using LC - MS and quantified using spiked non - yeast LCBs / ceramides as internal standard . Data are normalized to WT levels of the most abundant LCB / ceramide species ( set to 1 ) and presented as mean ± standard deviation from three independent experiments . Pairwise statistical significance was assessed by Student ' s t - test . p values are given above the respective bars .

O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The levels of the long chain bases (LCB) C18-dihydrosphingosine (C18-DHS) and C18-phytosphingosine (C18-PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆, Orm2-K25,33R, Orm2-3A and Orm2-3D mutants were measured using LC-MS and quantified using spiked non-yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.


( A ) Total cell lysates prepared from the indicated HCT116 cell lines were analyzed by immunoblotting . For comparison , different amounts of proteins ( 1 : 3 ratio ) were applied . An asterisk indicated non - specific crossreactive bands .

O O O O O O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Total cell lysates prepared from the indicated HCT116 cell lines were analyzed by immunoblotting. For comparison, different amounts of proteins (1:3 ratio) were applied. An asterisk indicated non-specific crossreactive bands.


( B ) The indicated cell lines were analyzed by immunofluorescence microscopy using anti - Cytochrome c antibody for mitochondria and anti - PMP70 antibody for peroxisome staining . Images are displayed as z - stacks of 6 confocal slices . Magnified images are also shown for mitochondrial morphologies . Scale bars , 10 μm . ( C ) Quantification of mitochondrial morphologies in ( B ) . Percentages of cells harboring fragmented , tubular , or elongated mitochondria are shown . Tubular and elongated denote normal tubular mitochondria seen in WT cells and highly connected mitochondrial network , respectively . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each of three replicate wells .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The indicated cell lines were analyzed by immunofluorescence microscopy using anti-Cytochrome c antibody for mitochondria and anti-PMP70 antibody for peroxisome staining. Images are displayed as z-stacks of 6 confocal slices. Magnified images are also shown for mitochondrial morphologies. Scale bars, 10 μm. (C) Quantification of mitochondrial morphologies in (B). Percentages of cells harboring fragmented, tubular, or elongated mitochondria are shown. Tubular and elongated denote normal tubular mitochondria seen in WT cells and highly connected mitochondrial network, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each of three replicate wells.


( A ) The indicated cell lines stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin for 3 hr and subjected to confocal immunofluorescence microscopy with anti - TOMM20 antibody . Scale bars , 10 μm . ( B ) Quantification of mCherry - Parkin translocation to mitochondria after 3 hr of valinomycin treatment . Partial or complete translocation to mitochondria in each cell was scored as separate phenotypes . Partial and complete denote that Parkin translocates to some of or all mitochondria , respectively . The error bars represent ± SD from three independent experiments . Over 100 cells were counted in each of three separate wells . ( C ) YFP - LC3 morphologies in ( A ) were quantified . Percentages of cells harboring diffuse , punctate or accumulated YFP - LC3 are shown . The error bars represent ± SD from three independent replicates . Over 100 cells were counted in each replicate . For the criteria of LC3 morphology , see Figure 2 - figure supplement 1 .

O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(A) The indicated cell lines stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to confocal immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial or complete translocation to mitochondria in each cell was scored as separate phenotypes. Partial and complete denote that Parkin translocates to some of or all mitochondria, respectively. The error bars represent ±SD from three independent experiments. Over 100 cells were counted in each of three separate wells. (C) YFP-LC3 morphologies in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate. For the criteria of LC3 morphology, see Figure 2-figure supplement 1.


( D ) YFP - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the absence or presence of HA - tagged TBC1D15 WT or Δ221 - 250 mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( E ) The YFP - LC3 morphology of cells in ( D ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the absence or presence of HA-tagged TBC1D15 WT or Δ221-250 mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (E) The YFP-LC3 morphology of cells in (D) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( F and G ) YFP - LC3 and mCherry - Parkin stably expressing WT ( F ) and TBC1D15 − / − ( G ) cells were treated with valinomycin for 3 hr and then subjected to immunoelectron microscopy with anti - GFP antibody . The square in panel a shows enlarged areas in panel b Scale bars , 500 nm .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(F and G) YFP-LC3 and mCherry-Parkin stably expressing WT (F) and TBC1D15−/− (G) cells were treated with valinomycin for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. The square in panel a shows enlarged areas in panel b Scale bars, 500 nm.


( A ) The indicated cells stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti - Cytochrome c antibody . Confocal images were acquired as z - stacks comprising 6 sequential sections with 0 . 8 μm z - intervals . Scale bars , 10 μm .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr followed by immunofluorescence microscopy with anti-Cytochrome c antibody. Confocal images were acquired as z-stacks comprising 6 sequential sections with 0.8 μm z-intervals. Scale bars, 10 μm.


( B ) TBC1D15 − / − cells prepared as in ( A ) were subjected to immunoelectron microscopy with anti - GFP antibody . White arrowheads indicate YFP - LC3 - labeled tubules . High magnification image is shown in the lower panel . Scale bars , 500 nm .

O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(B) TBC1D15−/− cells prepared as in (A) were subjected to immunoelectron microscopy with anti-GFP antibody. White arrowheads indicate YFP-LC3-labeled tubules. High magnification image is shown in the lower panel. Scale bars, 500 nm.


( C ) The indicated cells prepared as in ( A ) were subjected to immunostaining with anti - Tubulin antibody . YFP - LC3 and Tubulin staining are merged in the right panels . Magnified images are shown for TBC1D15 − / − cells . Scale bars , 10 μm .

O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(C) The indicated cells prepared as in (A) were subjected to immunostaining with anti-Tubulin antibody. YFP-LC3 and Tubulin staining are merged in the right panels. Magnified images are shown for TBC1D15−/− cells. Scale bars, 10 μm.


( D ) The indicated cells stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr . YFP - LC3 and mCherry - Parkin images are merged in the right panels . Scale bars , 10 μm . ( E ) YFP - LC3 morphologies of cells in ( D ) were quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) The indicated cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin in the presence or absence of nocodazole for 3 hr. YFP-LC3 and mCherry-Parkin images are merged in the right panels. Scale bars, 10 μm. (E) YFP-LC3 morphologies of cells in (D) were quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( A and B ) YFP - Parkin stably expressing cells were treated with valinomycin for indicated times . Total cell lysates were subjected to immunoblotting . I and II denote cytosolic and lipidated LC3B , respectively . An asterisk indicates ubiquitinated TOMM20 . ( C ) Indicated protein amounts as in ( B ) were quantified . The amount of protein without valinomycin treatment was set to 100 % . The error bars represent ± SD from three independent experiments .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(A and B) YFP-Parkin stably expressing cells were treated with valinomycin for indicated times. Total cell lysates were subjected to immunoblotting. I and II denote cytosolic and lipidated LC3B, respectively. An asterisk indicates ubiquitinated TOMM20. (C) Indicated protein amounts as in (B) were quantified. The amount of protein without valinomycin treatment was set to 100%. The error bars represent ±SD from three independent experiments.


( D ) YFP - Parkin stably expressing WT HCT116 cells were treated with or without valinomycin for 40 hr . Total cell lysates were analyzed by immunoblotting .

O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O

(D) YFP-Parkin stably expressing WT HCT116 cells were treated with or without valinomycin for 40 hr. Total cell lysates were analyzed by immunoblotting.


( A ) WT , MFF − / − or DRP1 − / − cells stably expressing YFP - LC3 and mCherry - Parkin were treated with valinomycin for 3 hr and subjected to immunofluorescence microscopy with anti - TOMM20 antibody . Scale bars , 10 μm . ( B ) Quantification of mCherry - Parkin translocation to mitochondria after 3 hr of valinomycin treatment . Partial and complete denote that Parkin translocates to some of and all mitochondria , respectively . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each replicate . ( C ) YFP - LC3 morphologies of cells in ( A ) were quantified . Percentages of cells harboring diffuse , punctate or accumulated YFP - LC3 are shown . The error bars represent ± SD from three independent replicates . Over 100 cells were counted in each replicate .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(A) WT, MFF−/− or DRP1−/− cells stably expressing YFP-LC3 and mCherry-Parkin were treated with valinomycin for 3 hr and subjected to immunofluorescence microscopy with anti-TOMM20 antibody. Scale bars, 10 μm. (B) Quantification of mCherry-Parkin translocation to mitochondria after 3 hr of valinomycin treatment. Partial and complete denote that Parkin translocates to some of and all mitochondria, respectively. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate. (C) YFP-LC3 morphologies of cells in (A) were quantified. Percentages of cells harboring diffuse, punctate or accumulated YFP-LC3 are shown. The error bars represent ±SD from three independent replicates. Over 100 cells were counted in each replicate.


( D ) WT , FIS1 − / − , and TBC1D15 − / − cells stably expressing YFP - LC3 were grown in starvation media . Z - stacks of confocal images are shown . Scale bars , 20 μm . ( E ) The number of YFP - LC3 dots in cells under growth or starvation conditions was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

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(D) WT, FIS1−/−, and TBC1D15−/− cells stably expressing YFP-LC3 were grown in starvation media. Z-stacks of confocal images are shown. Scale bars, 20 μm. (E) The number of YFP-LC3 dots in cells under growth or starvation conditions was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( F ) Total cell lysates from cells grown in normal or starvation media for 6 hr were subjected to immunoblotting . LC3 - I and LC3 - II denote cytosolic and lipidated LC3B , respectively .

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(F) Total cell lysates from cells grown in normal or starvation media for 6 hr were subjected to immunoblotting. LC3-I and LC3-II denote cytosolic and lipidated LC3B, respectively.


( A ) TBC1D15 − / − cells and those stably expressing HA - TBC1D15 WT and HA - TBC1D15 ( Δ221 - 250 ) , and FIS1 − / − cells stably expressing HA - TBC1D15 WT were subjected to immunostaining with anti - TOMM20 and anti - HA antibodies . Scale bars , 10 μm .

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(A) TBC1D15−/− cells and those stably expressing HA-TBC1D15 WT and HA-TBC1D15 (Δ221-250), and FIS1−/− cells stably expressing HA-TBC1D15 WT were subjected to immunostaining with anti-TOMM20 and anti-HA antibodies. Scale bars, 10 μm.


( B ) HA - TBC1D15 WT or HA - TBC1D15 ( Δ221 - 250 ) with or without Fis1 was transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - HA and anti - Fis1 antibodies . Scale bars , 20 μm .

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(B) HA-TBC1D15 WT or HA-TBC1D15 (Δ221-250) with or without Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Scale bars, 20 μm.


( C ) YFP - TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - Fis1 and anti - Cytochrome c antibodies . Scale bars , 20 μm .

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(C) YFP-TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-Fis1 and anti-Cytochrome c antibodies. Scale bars, 20 μm.


( D ) YFP or YFP - Fis1 was co - overexpressed with HA - TBC1D15 or HA - TBC1D17 in HEK293 cells . The cell extracts were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) and anti - GFP ( lower panel ) antibodies .

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(D) YFP or YFP-Fis1 was co-overexpressed with HA-TBC1D15 or HA-TBC1D17 in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.


( E ) YFP , YFP - TBC1D15 , or YFP - TBC1D17 was co - overexpressed with HA - TBC1D15 ( upper panel ) or HA - TBC1D17 ( lower panel ) in HEK293 cells . The cell extracts were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP and anti - HA antibodies .

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(E) YFP, YFP-TBC1D15, or YFP-TBC1D17 was co-overexpressed with HA-TBC1D15 (upper panel) or HA-TBC1D17 (lower panel) in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP and anti-HA antibodies.


( F ) HA - TBC1D15 ( Δ221 - 250 ) together with Fis1 and YFP - TBC1D15 WT or YFP - TBC1D17 WT were transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - HA and anti - Fis1 antibodies . Images of HA and Fis1 staining were merged in the right panels . Scale bars , 20 μm .

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(F) HA-TBC1D15 (Δ221-250) together with Fis1 and YFP-TBC1D15 WT or YFP-TBC1D17 WT were transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Images of HA and Fis1 staining were merged in the right panels. Scale bars, 20 μm.


( A ) The indicated cells transiently expressing YFP - GABARAPL1 ( green ) and mCherry - Parkin ( red ) were treated with valinomycin for 3 hr . Scale bars , 10 μm .

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(A) The indicated cells transiently expressing YFP-GABARAPL1 (green) and mCherry-Parkin (red) were treated with valinomycin for 3 hr. Scale bars, 10 μm.


( B ) YFP - TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST - fused proteins ( GABA , L1 , and L2 represent GABARAP , GABARAPL1 , and GABARAPL2 , respectively ) . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) . Coomassie brilliant blue ( CBB ) staining shows GST - fusion proteins in bound fractions ( lower panel ) . ( C ) Binding assay carried out as in ( B ) with GST - GABARAPL1 WT ( GST - WT ) or its Y49A / L50A mutant ( GST - YL ) . Immunoblotting with anti - GFP antibody ( upper panel ) and CBB staining ( lower panel ) are shown .

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(B) YFP-TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST-fused proteins (GABA, L1, and L2 represent GABARAP, GABARAPL1, and GABARAPL2, respectively). 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel). Coomassie brilliant blue (CBB) staining shows GST-fusion proteins in bound fractions (lower panel). (C) Binding assay carried out as in (B) with GST-GABARAPL1 WT (GST-WT) or its Y49A/L50A mutant (GST-YL). Immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel) are shown.


( D ) Cell extracts from HEK293 overexpressed HA - TBC1D15 and YFP , YFP - GABARAPL1 ( YFP - WT ) , or its Y49AL50A mutant ( YFP - YL ) were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) and anti - GFP ( lower panel ) antibodies .

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(D) Cell extracts from HEK293 overexpressed HA-TBC1D15 and YFP, YFP-GABARAPL1 (YFP-WT), or its Y49AL50A mutant (YFP-YL) were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.


( E - H ) The indicated YFP - tagged TBC1D15 full - length , truncated , or point - mutant protein or YFP - Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST - GABARAPL1 . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) and CBB staining ( lower panel ) .

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(E-H) The indicated YFP-tagged TBC1D15 full-length, truncated, or point-mutant protein or YFP-Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST-GABARAPL1. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel).


( J ) LC3 - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the presence of HA - tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( K ) The YFP - LC3 morphology of cells in ( J ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

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(J) LC3-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( A ) Total cell lysates from HCT116 treated with control ( NTC ) or RAB7A siRNA were analyzed by immunoblotting .

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(A) Total cell lysates from HCT116 treated with control (NTC) or RAB7A siRNA were analyzed by immunoblotting.


( B ) The indicated cells stably expressing YFP - LC3 ( green ) and mCherry - Parkin were treated with control ( NTC ) or Rab7 _ # 5 siRNA . After 3 hr valinomycin treatment , cells were analyzed by immunofluorescence microscopy with anti - TOMM20 antibody ( red ) . Z - stacks of confocal images are shown . Magnified images are also shown . Scale bars , 10 μm . ( C ) YFP - LC3 morphologies of cells in ( B ) were quantified . Percentages of cells harboring diffuse , punctuate or accumulated / tubulated YFP - LC3 are shown . Data and error bars were obtained from at least 50 cells in each of three independent replicates .

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(B) The indicated cells stably expressing YFP-LC3 (green) and mCherry-Parkin were treated with control (NTC) or Rab7_#5 siRNA. After 3 hr valinomycin treatment, cells were analyzed by immunofluorescence microscopy with anti-TOMM20 antibody (red). Z-stacks of confocal images are shown. Magnified images are also shown. Scale bars, 10 μm. (C) YFP-LC3 morphologies of cells in (B) were quantified. Percentages of cells harboring diffuse, punctuate or accumulated/tubulated YFP-LC3 are shown. Data and error bars were obtained from at least 50 cells in each of three independent replicates.


( D ) The indicated cells stably expressing YFP - LC3 ( green ) , mCherry - Parkin , and 2HA - Rab7 ( Red ) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti - HA antibody . Magnified images are also shown . Scale bars , 10 μm .

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(D) The indicated cells stably expressing YFP-LC3 (green), mCherry-Parkin, and 2HA-Rab7 (Red) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti-HA antibody. Magnified images are also shown. Scale bars, 10 μm.


( E ) YFP - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the presence of HA - tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( F ) The YFP - LC3 morphology of cells in ( E ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each replicate .

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(E) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (F) The YFP-LC3 morphology of cells in (E) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate.


( b ) Effects of C18 - Pyr - Cer ( C18PC , lanes 2 and 4 ) on the lipidation of LC3B ( LC3B - II ) were examined by western blotting in the absence or presence of a pan - caspase inhibitor Z - VAD ( lanes 3 and 4 ) and compared to vehicle - treated controls ( lanes 1 and 3 ) . Full blots can be found in Supplementary Figure 13 .

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(b) Effects of C18-Pyr-Cer (C18PC, lanes 2 and 4) on the lipidation of LC3B (LC3B-II) were examined by western blotting in the absence or presence of a pan-caspase inhibitor Z-VAD (lanes 3 and 4) and compared to vehicle-treated controls (lanes 1 and 3). Full blots can be found in Supplementary Figure 13.


( c ) Formation of double - membrane autophagosomal vesicles ( black arrows ) in the absence ( control ) or presence of C18PC was visualized by TEM ( left and right , respectively ) . Higher magnification of TEM visualization is shown in lower panels . Scale bars , 10 μm ( top ) and 500 nm ( bottom ) .

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(c) Formation of double-membrane autophagosomal vesicles (black arrows) in the absence (control) or presence of C18PC was visualized by TEM (left and right, respectively). Higher magnification of TEM visualization is shown in lower panels. Scale bars, 10 μm (top) and 500 nm (bottom).


( d ) Effects of siRNA - mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18PC were determined and compared to effects of transfection with Scr siRNAs ( controls ) using Trypan blue exclusion assay .

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(d) Effects of siRNA-mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18PC were determined and compared to effects of transfection with Scr siRNAs (controls) using Trypan blue exclusion assay.


( e ) Roles of C18PC in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild - type ( ATG5 + / + ) and ATG5 − / − knockout mice with C18PC . Control cells were treated with vehicle .

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(e) Roles of C18PC in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild-type (ATG5+/+) and ATG5−/− knockout mice with C18PC. Control cells were treated with vehicle.


( f ) Effects of C18PC on caspase - dependent or caspase - independent cell death were determined in MEFs isolated from Bax − / − Bak − / − or Casp3 − / − Casp7 − / − mice and from wild - type ( Bax + / + Bak + / + ) or Casp3 + / − Casp7 + / − mice , used as controls . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(f) Effects of C18PC on caspase-dependent or caspase-independent cell death were determined in MEFs isolated from Bax−/− Bak−/− or Casp3−/− Casp7−/− mice and from wild-type (Bax+/+ Bak+/+) or Casp3+/− Casp7+/− mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( a ) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18 - Pyr - Cer ( C18PC ) at 2 h in UM - SCC22A ( 22A ) cells was examined by visualizing the colocalization of MTG ( green ) and LTR ( red ) using live - cell imaging and confocal microscopy . Scale bars , 10 μm . Enlarged images are shown in boxed areas .

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(a) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18-Pyr-Cer (C18PC) at 2 h in UM-SCC22A (22A) cells was examined by visualizing the colocalization of MTG (green) and LTR (red) using live-cell imaging and confocal microscopy. Scale bars, 10 μm. Enlarged images are shown in boxed areas.


( b ) Effects of C18 - dihydro - Cer - 14 - piperidine ( dihydro - C18PC ) on OCR measured using the SeaHorse alongside those of C18PC - and vehicle - treated controls . Data shown are an average of at least two independent experiments performed in duplicate ± s . d . ( * P 0 . 05 ) .

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(b) Effects of C18-dihydro-Cer-14-piperidine (dihydro-C18PC) on OCR measured using the SeaHorse alongside those of C18PC- and vehicle-treated controls. Data shown are an average of at least two independent experiments performed in duplicate ± s.d. (*P 0.05).


( a ) Effects of expression of wild - type ( WT ) CerS1 and its catalytically inactive V5 - tagged mutant ( Mut ) , which cannot generate C18 - ceramide , on the formation of LC3B - II were determined by western blotting ( + / − tet ) . Successful induction of wild - type and mutant CerS1 expression was confirmed using anti - V5 . β - actin was used as a loading control . Full blots can be found in Supplementary Figure 13 .

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(a) Effects of expression of wild-type (WT) CerS1 and its catalytically inactive V5-tagged mutant (Mut), which cannot generate C18-ceramide, on the formation of LC3B-II were determined by western blotting (+/− tet). Successful induction of wild-type and mutant CerS1 expression was confirmed using anti-V5. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.


( b ) Generation of endogenous C18 - and C18 : 1 - ceramides in response to wild - type CerS1 and mutant CerS1 induction was measured by LC / MS / MS .

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(b) Generation of endogenous C18- and C18:1-ceramides in response to wild-type CerS1 and mutant CerS1 induction was measured by LC/MS/MS.


( c ) Effects of wild - type CerS1 and C18 - ceramide induction ( + tet ) on GFP and LC3B - GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls ( − tet ) . Scale bars , 10

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(c) Effects of wild-type CerS1 and C18-ceramide induction (+ tet) on GFP and LC3B-GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls (− tet). Scale bars, 10 μm


( d ) The effects of induction of wild - type CerS1 and C18 - ceramide and catalytically inactive mutant CerS1 ( + tet ) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls ( − tet ) .

O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

(d) The effects of induction of wild-type CerS1 and C18-ceramide and catalytically inactive mutant CerS1 (+ tet) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls (− tet).


( e ) Targeting mitochondria with autophagolysosomes in the absence ( − tet ) or presence ( + tet ) of wild - type CerS1 and C18 - ceramide was visualized by colocalization of MTG and LTR using confocal microscopy . Scale bars , 10 μm .

O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(e) Targeting mitochondria with autophagolysosomes in the absence (− tet) or presence (+ tet) of wild-type CerS1 and C18-ceramide was visualized by colocalization of MTG and LTR using confocal microscopy. Scale bars, 10 μm.


( f ) Effects of wild - type and mutant CerS1 ( − / + tet ) on ATP generation .

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(f) Effects of wild-type and mutant CerS1 (−/+ tet) on ATP generation.


( g ) Effects of wild - type CerS1 ( − / + tet ) on ATP generation in the absence ( control shRNA ) or presence of shRNA - mediated knockdown of LC3B . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(g) Effects of wild-type CerS1 (−/+ tet) on ATP generation in the absence (control shRNA) or presence of shRNA-mediated knockdown of LC3B. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( a ) Subcellular localization of ceramide , generated by wild - type CerS1 induction ( + tet ) , was visualized in mitochondria by colocalization of ceramides and mitochondria with anti - ceramide and anti - Tom20 , respectively , using confocal microscopy .

O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O

(a) Subcellular localization of ceramide, generated by wild-type CerS1 induction (+ tet), was visualized in mitochondria by colocalization of ceramides and mitochondria with anti-ceramide and anti-Tom20, respectively, using confocal microscopy.


( b , c ) Mitochondrial ( Mito ) targeting of LC3B - II and C18 - ceramide , generated by induction of wild - type CerS1 ( b ) , and that of the products of the catalytically inactive mutant CerS1 ( + tet ) ( c ) was visualized by colocalization of ceramide ( Cer ; green ) , CFP - LC3B ( blue ) and MTR using anti - ceramide and confocal microscopy . Noninduced cells ( − tet ) were used as controls . Scale bars , 10 μm .

O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(b,c) Mitochondrial (Mito) targeting of LC3B-II and C18-ceramide, generated by induction of wild-type CerS1 (b), and that of the products of the catalytically inactive mutant CerS1 (+ tet) (c) was visualized by colocalization of ceramide (Cer; green), CFP-LC3B (blue) and MTR using anti-ceramide and confocal microscopy. Noninduced cells (− tet) were used as controls. Scale bars, 10 μm.


( a ) Binding of Flag - tagged wild - type LC3B and LC3BI35A , LC3BF52A or LC3BG120A with endogenous C18 - ceramide ( C18 - Cer ) , generated in response to wild - type CerS1 induction ( left ) or exogenous C18 - Pyr - Cer ( right ) , was measured using LC / MS / MS after pulldown using anti - Flag - conjugated beads . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Binding of Flag-tagged wild-type LC3B and LC3BI35A, LC3BF52A or LC3BG120A with endogenous C18-ceramide (C18-Cer), generated in response to wild-type CerS1 induction (left) or exogenous C18-Pyr-Cer (right), was measured using LC/MS/MS after pulldown using anti-Flag-conjugated beads. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( b ) Effects of CerS1 and C18 - ceramide induction on the lipidation of Flag - tagged wild - type , LC3BI35A , LC3BF52A and LC3BG120A proteins were examined by western blotting . Noninduced cells ( − tet ) were used as controls . β - actin was used as a loading control . Full blots can be found in Supplementary Figure 13 .

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(b) Effects of CerS1 and C18-ceramide induction on the lipidation of Flag-tagged wild-type, LC3BI35A, LC3BF52A and LC3BG120A proteins were examined by western blotting. Noninduced cells (− tet) were used as controls. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.


( a , b ) Effects of G120A and F52A mutations , which perturbed C18 - ceramide binding of LC3B , on targeting of mitochondria by LC3B - containing autophagosomes ( visualized by confocal microscopy using anti - Flag and anti - Tom20 ) ( a ) or mitochondrial function ( measurement of OCR ) ( b ) . V , vector .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O

(a,b) Effects of G120A and F52A mutations, which perturbed C18-ceramide binding of LC3B, on targeting of mitochondria by LC3B-containing autophagosomes (visualized by confocal microscopy using anti-Flag and anti-Tom20) (a) or mitochondrial function (measurement of OCR) (b). V, vector.


( c ) Effects of siRNA - mediated knockdown of p62 , NIX and Drp1 on OCR in the absence or presence of CerS1 and C18 - ceramide induction were measured using the SeaHorse . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(c) Effects of siRNA-mediated knockdown of p62, NIX and Drp1 on OCR in the absence or presence of CerS1 and C18-ceramide induction were measured using the SeaHorse. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( d ) Effects of siRNA - mediated knockdown of Drp1 ( lower left and right ) on the localization of ceramide within mitochondrial membranes were visualized using the colocalization of anti - ceramide ( red ) and anti - Tom20 ( green ) under confocal microscopy , in the absence ( upper and lower left ) or presence ( upper and lower right ) of CerS1 and C18 - ceramide induction ( − / + tet ) compared to Scr siRNA - transfected controls ( upper left and right ) . Scale bars , 10 μm in a and d . Enlarged images are shown in boxed areas in d .

O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Effects of siRNA-mediated knockdown of Drp1 (lower left and right) on the localization of ceramide within mitochondrial membranes were visualized using the colocalization of anti-ceramide (red) and anti-Tom20 (green) under confocal microscopy, in the absence (upper and lower left) or presence (upper and lower right) of CerS1 and C18-ceramide induction (−/+ tet) compared to Scr siRNA-transfected controls (upper left and right). Scale bars, 10 μm in a and d. Enlarged images are shown in boxed areas in d.


( a , b ) Roles of shRNA - mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function ( a ) or UM - SCC - 22A xenograft - derived tumor growth ( b ) in the absence or presence of wild - type CerS1 and C18 - ceramide induction ( − / + tet ) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice ( n = 6 per group ) , respectively . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(a,b) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a) or UM-SCC-22A xenograft-derived tumor growth (b) in the absence or presence of wild-type CerS1 and C18-ceramide induction (−/+ tet) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice (n = 6 per group), respectively. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


Representative confocal images of control ( CTL - 1 , 2 , 3 ) and DOA + patient fibroblasts carrying indicated monoallelic mutations imaged as described in A . Scale bar = 20μm . Passage number between P12 - 15 . Mitochondrial morphology quantification of B . Data represent mean ± SD of two independent experiments , ( 195 - 2496 cells per cell line ) , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not

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Representative confocal images of control (CTL-1, 2, 3) and DOA+ patient fibroblasts carrying indicated monoallelic mutations imaged as described in A. Scale bar=20μm. Passage number between P12-15. Mitochondrial morphology quantification of B. Data represent mean ± SD of two independent experiments, (195-2496 cells per cell line), One-way ANOVA; ** p < 0.01, ****p < 0.0001, ns; not significant


Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with OPA1 , DNM1L , or non - targeting ( NT ) siRNAs for 72 hours and imaged as described in A . Scale bar = 20μm . Passage number between P12 - 14 . Mitochondrial morphology quantification of D . Data represent mean ± SD of three independent experiments , ( 3219 - 5857 cells per cell line ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, or non-targeting (NT) siRNAs for 72 hours and imaged as described in A. Scale bar=20μm. Passage number between P12-14. Mitochondrial morphology quantification of D. Data represent mean ± SD of three independent experiments, (3219-5857 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.


Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide ( CHX ) where indicated for 6 hours . Imaging as described in A . Scale bar = 20μm . Passage number between P14 - P15 . Mitochondrial morphology quantification of F . Data represent mean ± SD of two independent experiments , ( 879 - 4154 cells per cell line ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm. Passage number between P14-P15. Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.


Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with OPA1 , DNM1L , PGS1 , and non - targeting ( NT ) siRNAs or indicated combinations for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 20μm . Passages number between P10 - 15 . Mitochondrial morphology quantification of ( A ) using control fibroblasts with fragmented ( OPA1 siRNA ) , normal ( non - targeting NT siRNA ) , and hypertubulated ( DNM1L siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 905 - 3695 cells per cell line ) , ( % fragmented ) ; ; * * * * p < 0 . 0001 , ns ; not significant . .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD B-ORGANISM B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. Passages number between P10-15. Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented (OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubulated (DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented); ; ****p < 0.0001, ns; not significant..


Representative confocal images of wild type ( WT ) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours . Live imaging of mitochondria ( mitoYFP , green ) and nuclei ( NucBlue , blue ) . Scale bar = 10μm . Mitochondrial morphology quantification of ( C ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) ground truth training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 6613 - 8758 cells per cell line ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of wild type (WT) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant.


Representative confocal images of WT , Opa1Crispr MEFs complemented with pLenti - Opa1 , Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti - Pgs1 by lentiviral delivery . Live imaging of mitochondria ( mitoYFP , green ) and nuclei ( NucBlue , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( E ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 691 - 3990 cells per cell line ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant . .

O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-GENEPROD B-CELL_TYPE O O O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of WT, Opa1Crispr MEFs complemented with pLenti-Opa1, Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant..


Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m - chlorophenyl hydrazine ( CCCP ) for the indicated time points . Images were captured every hour for 18 hours . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of three independent experiments , ( 131 - 426 cells per cell line ) , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for the indicated time points. Images were captured every hour for 18 hours. Scale bar=10μm. Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of three independent experiments, (131-426 cells per cell line), One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion ( SiMH ) with 10μM cycloheximide ( CHX ) for the indicated time points . Images were captured every hour for 9 hours . Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of four independent experiments , ( 155 - 745 cells per cell line ) , One - way ANOVA .

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion (SiMH) with 10μM cycloheximide (CHX) for the indicated time points. Images were captured every hour for 9 hours. Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of four independent experiments, (155-745 cells per cell line), One-way ANOVA.


Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( B ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , and hypertubular ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 726 - 4236 cells per cell line ) , ( % fragmented ) ; * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant .

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular (Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented); *** p < 0.001, ****p < 0.0001, ns; not significant.


Representative confocal micrographs of MEFs WT , Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( G ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , and hypertubulated ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of > 3 independent experiments , ( 3096 - 7238 cells per cell line ) , One - way ANOVA ( % fragmented ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal micrographs of MEFs WT, Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubulated (Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented) ; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs . Scale bar = 200nm . Quantification of ( C ) of mitochondrial ultrastructure ; outer membrane / inner membrane ration ( IMM / OMM ) and cristae number per mitochondrion . Violin plot of > 50 mitochondria per cell line , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs. Scale bar=200nm. Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (IMM/OMM) and cristae number per mitochondrion. Violin plot of >50 mitochondria per cell line, One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


mtDNA content in MEFs from ( F ) was quantified by amplification of MTTL1 , 16S and MT - ND1 genes relative to the GAPDH nuclear gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant . mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1 , 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mtDNA content in MEFs from (F) was quantified by amplification of MTTL1, 16S and MT-ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; ****p < 0.0001, ns; not significant. mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1, 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ****p < 0.0001, ns; not significant.


( G ) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS - PAGE ( horizontal line denotes separate membranes ) , immunoblotted with indicated antibodies and quantified by densitometry ( H ) . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant .

O O O O O O O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS-PAGE (horizontal line denotes separate membranes), immunoblotted with indicated antibodies and quantified by densitometry (H). Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.


A , Schematic representation of signaling events . Left : time course of changes in cAMP ( dotted line ) and cGMP ( solid line ) ; right : time course of changes in Vm ( green ) , pHi ( red ) ; [ Na + ] i ( orange ) , and [ Ca2 + ] i ( pink ) . Vertical dotted lines indicate time of stimulation .

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A, Schematic representation of signaling events. Left: time course of changes in cAMP (dotted line) and cGMP (solid line); right: time course of changes in Vm (green), pHi (red); [Na+]i (orange), and [Ca2+]i (pink). Vertical dotted lines indicate time of stimulation.


A - D , Longitudinal ( A and C , proximal view on left ) and cross - sectional ( B and D , proximal - to - distal view ) tomographic slices of representative intact flagella from wild - type Chlamydomonas reinhardtii ( C . r . ) flagella ( A and B ) and sea urchin Strongylocentrotus purpuratus ( S . p . ) sperm ( C and D ) . Note that in Chlamydomonas , the flagellar membrane ( m ) is surrounded by a glycocalyx ( g ) . Other labels : central pair complex ( c ) and doublet microtubule ( d ) . Scale bar , 100 nm .

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A-D, Longitudinal (A and C, proximal view on left) and cross-sectional (B and D, proximal-to-distal view) tomographic slices of representative intact flagella from wild-type Chlamydomonas reinhardtii (C.r.) flagella (A and B) and sea urchin Strongylocentrotus purpuratus (S.p.) sperm (C and D). Note that in Chlamydomonas, the flagellar membrane (m) is surrounded by a glycocalyx (g). Other labels: central pair complex (c) and doublet microtubule (d). Scale bar, 100 nm.


E , Diameter of intact flagella from Chlamydomonas and sea urchin sperm measured on cross - sectional views of cryo - tomographic reconstructions . Numbers indicate average diameters ± s . d . ( n = 84 for sea urchin ( S . p . ) and n = 18 for Chlamydomonas ( C . r . ) )

O O B-EXP_ASSAY O O B-SUBCELLULAR O B-ORGANISM O B-ORGANISM I-ORGANISM B-CELL_TYPE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O

E, Diameter of intact flagella from Chlamydomonas and sea urchin sperm measured on cross-sectional views of cryo-tomographic reconstructions. Numbers indicate average diameters ± s.d. (n = 84 for sea urchin (S.p.) and n = 18 for Chlamydomonas (C.r.))


A , Time course of the change in voltage Vm evoked by a 2 - ms light flash in sperm that was loaded with caged cGMP ( 15 μM ) and recorded in 100KASW . Inset : extended timescale showing the rise of Vm . Fit of data ( black ) using two exponentials with time constants τrise = 17 . 4 ms and τrelax = 354 ms . Light flash in all panels is depicted as dashed magenta line .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Time course of the change in voltage Vm evoked by a 2-ms light flash in sperm that was loaded with caged cGMP (15 μM) and recorded in 100KASW. Inset: extended timescale showing the rise of Vm. Fit of data (black) using two exponentials with time constants τrise = 17.4 ms and τrelax = 354 ms. Light flash in all panels is depicted as dashed magenta line.


B , Time course of the changes in Vm evoked by 100 - ms flash in 100KASW using caged cGMP ( blue ) and caged 8 - Br - cGMP ( black ) . The flashes released 260 nM cGMP or 2 , 600 nM 8 - Br - cGMP .

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B, Time course of the changes in Vm evoked by 100-ms flash in 100KASW using caged cGMP (blue) and caged 8-Br-cGMP (black). The flashes released 260 nM cGMP or 2,600 nM 8-Br-cGMP.


C , Time course of changes in Vm in 100KASW evoked by 2 - s flashes at different light energy that correspond to 440 , 220 , 75 , and 8 nM s - 1 of cGMP . Data was fitted using a heuristic model described by equation 3 ( Methods section ) ( black lines ) . D , Amplitude ( red ) and decay time τrelax ( black ) for different light energies and release rates for experiments as shown in panel C . τrelax was determined using a single exponential fit . The linear fit of the Vm amplitude was restricted to low light levels ; at higher levels , the Vm response saturates ( see panel C ) . Points indicate mean ± s . d . from 5 experiments .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

C, Time course of changes in Vm in 100KASW evoked by 2-s flashes at different light energy that correspond to 440, 220, 75, and 8 nM s-1 of cGMP. Data was fitted using a heuristic model described by equation 3 (Methods section) (black lines). D, Amplitude (red) and decay time τrelax (black) for different light energies and release rates for experiments as shown in panel C. τrelax was determined using a single exponential fit. The linear fit of the Vm amplitude was restricted to low light levels; at higher levels, the Vm response saturates (see panel C). Points indicate mean ± s.d. from 5 experiments.


A , Time course of cGMP - evoked changes in Vm in 100KASW ( red , 500 nM cGMP ; pink , 9 nM cGMP ) and normal ASW ( blue , 500 nM cGMP ; navy , 9 nM cGMP ) . The time constants of recovery after cGMP release ( 9 nM ) were 289 ms ( depolarization ) and 303 ms ( hyperpolarization ) . Signals in panels were detected using the potentiometric probe FluoVolt ; dashed magenta lines indicate the timing of the photolysis flash .

O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O

A, Time course of cGMP-evoked changes in Vm in 100KASW (red, 500 nM cGMP; pink, 9 nM cGMP) and normal ASW (blue, 500 nM cGMP; navy, 9 nM cGMP ). The time constants of recovery after cGMP release (9 nM) were 289 ms (depolarization) and 303 ms (hyperpolarization). Signals in panels were detected using the potentiometric probe FluoVolt; dashed magenta lines indicate the timing of the photolysis flash.


, Representative measurement of the dependence of recovery - time constant on the amount of released cGMP . Inset : decay time was constant for low stimulation levels equivalent to ≲ 30 cGMP molecules or ≤ 3 resact molecules .

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, Representative measurement of the dependence of recovery-time constant on the amount of released cGMP. Inset: decay time was constant for low stimulation levels equivalent to ≲ 30 cGMP molecules or ≤ 3 resact molecules.


, Voltage response evoked by photolysis of DEACM - caged cGMP ( blue ) or 8 - Br - cGMP ( red ) . The light energy was adjusted such that Vm response amplitudes became similar ; for 8 - Br - cGMP , about 5 - times more light energy was required . Light pulse 10 ms .

O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

, Voltage response evoked by photolysis of DEACM-caged cGMP (blue) or 8-Br-cGMP (red). The light energy was adjusted such that Vm response amplitudes became similar; for 8-Br-cGMP, about 5-times more light energy was required. Light pulse 10 ms.


, Vm responses evoked by cGMP in the presence ( blue ) and absence ( red ) of the HCN channel blocker ZD7288 ( 50 μM ) .

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, Vm responses evoked by cGMP in the presence (blue) and absence (red) of the HCN channel blocker ZD7288 (50 μM).


E , cGMP - evoked Vm response in ASW ( black ) , in the presence of IBMX ( 1 mM ) ( red ) , and in Ca2 + - free ASW ( [ Ca2 + ] ≤ 500 nM ) in the presence of IBMX ( 1 mM ) ( blue ) . Signals were detected using the potentiometric probe Di - 8 - ANEPPS in the ratio mode . F , Normalized responses from panel E .

O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

E, cGMP-evoked Vm response in ASW (black), in the presence of IBMX (1 mM) (red), and in Ca2+-free ASW ([Ca2+] ≤ 500 nM) in the presence of IBMX (1 mM) (blue). Signals were detected using the potentiometric probe Di-8-ANEPPS in the ratio mode. F, Normalized responses from panel E.


A , B , Ca2 + signals elicited by cGMP release and recorded with FluoForte . The UV flash released 28 ( A ) and 215 B ) cGMP molecules . Light flashes are indicated by dashed magenta lines .

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A, B, Ca2+ signals elicited by cGMP release and recorded with FluoForte. The UV flash released 28 (A) and 215 B) cGMP molecules. Light flashes are indicated by dashed magenta lines.


C , pHi signals , elicited by the release of approximately 1 , 400 cGMP molecules , were recorded with pHrodo Red . An increase of pHi is indicated by a decrease of ∆ F / F . Exemplary recording from a single cell . D , Average of pHi signals recorded from n = 25 cells . Error bars represent s . d .

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C, pHi signals, elicited by the release of approximately 1,400 cGMP molecules, were recorded with pHrodo Red. An increase of pHi is indicated by a decrease of ∆F/F. Exemplary recording from a single cell. D, Average of pHi signals recorded from n = 25 cells. Error bars represent s.d.


B , Relationship between cGMP synthesis rate and free cGMP concentration . We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset : expanded scale .

O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

B, Relationship between cGMP synthesis rate and free cGMP concentration. We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset: expanded scale.


B ) Body weights of E18 . 5 wild - type , TKO and QKO foetuses . Data for individual animals are presented as circles with mean ± SEM . Data were analysed by one - way ANOVA followed by multiple comparison with Benjamini and Hochberg correction .

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B) Body weights of E18.5 wild-type, TKO and QKO foetuses. Data for individual animals are presented as circles with mean ± SEM. Data were analysed by one-way ANOVA followed by multiple comparison with Benjamini and Hochberg correction.


C ) Percentages of E18 . 5 QKO foetuses exhibiting the indicated externally visible developmental defects ( numbers above bars represent the number of animals examined ) . Age - matched TKO animals are included for comparison . An example of a QKO E18 . 5 pup presenting with an omphalocele ( asterisk ) , exencephaly ( arrow ) , and spina bifida ( arrowhead ) is shown alongside an age - matched WT control . Scale bar = 5 mm .

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C) Percentages of E18.5 QKO foetuses exhibiting the indicated externally visible developmental defects (numbers above bars represent the number of animals examined). Age-matched TKO animals are included for comparison. An example of a QKO E18.5 pup presenting with an omphalocele (asterisk), exencephaly (arrow), and spina bifida (arrowhead) is shown alongside an age-matched WT control. Scale bar = 5 mm.


A ) Percentages of E18 . 5 QKO foetuses exhibiting the indicated defects when examined under a dissection microscope . Age - matched TKO animals are included for comparison . Numbers above bars represent number of animals examined .

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A) Percentages of E18.5 QKO foetuses exhibiting the indicated defects when examined under a dissection microscope. Age-matched TKO animals are included for comparison. Numbers above bars represent number of animals examined.


B - D ) Images depicting the hindlimbs ( HL ) , tail ( T ) , forelimbs ( FL ) , and ventral view of the palate ( P ) in a wild - type foetus at E18 . 5 of development . E - G ) Corresponding images of a representative age - matched QKO foetus . Panel G shows a cleft palate ( indicated by arrowheads ) . H , I ) Magnified images of panels E and F . Arrows indicate excess tissue at the anterior and posterior edges of the hindlimb and forelimb , respectively , of QKO foetuses .

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B-D) Images depicting the hindlimbs (HL), tail (T), forelimbs (FL), and ventral view of the palate (P) in a wild-type foetus at E18.5 of development. E-G) Corresponding images of a representative age-matched QKO foetus. Panel G shows a cleft palate (indicated by arrowheads). H, I) Magnified images of panels E and F. Arrows indicate excess tissue at the anterior and posterior edges of the hindlimb and forelimb, respectively, of QKO foetuses.


J ) Percentages of QKO animals exhibiting various aortic arch abnormalities and great vessel defects ( n = 19 ) .

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J) Percentages of QKO animals exhibiting various aortic arch abnormalities and great vessel defects (n = 19).


K ) Aortic arch and vessels in a wild - type foetus . A corresponding drawing to explain the images is shown on the right .

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K) Aortic arch and vessels in a wild-type foetus. A corresponding drawing to explain the images is shown on the right.


L , M ) Images of QKO foetuses and accompanying illustration of vessel defects . Absence of the right subclavian artery ( L ) and persistent truncus arteriosus ( pTA ) with a right curving pulmonary trunk / ductus arteriosus ( PT / rcDA ; M ) .

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L, M) Images of QKO foetuses and accompanying illustration of vessel defects. Absence of the right subclavian artery (L) and persistent truncus arteriosus (pTA) with a right curving pulmonary trunk / ductus arteriosus (PT/rcDA; M).


A - C ) Heads of E18 . 5 foetuses were serially sectioned in coronal orientation and stained with H & E . Representative images of rostro - occipital level - matched sections from age - matched wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( TKO ; n = 4 ) and QKO foetuses ( n = 4 ) depicting the forebrain , nasal cavity , and tongue are shown . BP , bony palate ; Cx , cerebral cortex ; ES , epidural space ; In , incisor ; LV ; lateral ventricle ; Ma , mandible ; MC , mouth cavity ; NC , nasal cavity ; NS , nasal septum ; St , striatum ; SVZ , subventricular zone ; To , tongue ; WP , whisker follicles . Scale bar = 850 µm ( A ) , 1200 µm ( B , C ) .

O O O O B-TISSUE O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O

A-C) Heads of E18.5 foetuses were serially sectioned in coronal orientation and stained with H&E. Representative images of rostro-occipital level-matched sections from age-matched wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (TKO; n = 4) and QKO foetuses (n = 4) depicting the forebrain, nasal cavity, and tongue are shown. BP, bony palate; Cx, cerebral cortex; ES, epidural space; In, incisor; LV; lateral ventricle; Ma, mandible; MC, mouth cavity; NC, nasal cavity; NS, nasal septum; St, striatum; SVZ, subventricular zone; To, tongue; WP, whisker follicles. Scale bar = 850 µm (A), 1200 µm (B, C).


D ) Morphometric assessment of indicated tissue areas . Data of individual animals are displayed as circles with means and standard error of the mean . Data were analysed by unpaired , two - tailed Student ' s t - test . Related data normalised to mean bodyweight are displayed in Figure EV1 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Morphometric assessment of indicated tissue areas. Data of individual animals are displayed as circles with means and standard error of the mean. Data were analysed by unpaired, two-tailed Student's t-test. Related data normalised to mean bodyweight are displayed in Figure EV1.


A - C ) Representative H & E - stained coronal serial sections of the eye from E18 . 5 wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( n = 3 ) , and Bok - / - Bak - / - Bax - / - Bid - / - quadruple knockout ( n = 3 ) foetuses .

O O O O O O O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-TISSUE O

A-C) Representative H&E-stained coronal serial sections of the eye from E18.5 wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (n = 3), and Bok-/-Bak-/-Bax-/-Bid-/- quadruple knockout (n = 3) foetuses.


D ) Densitometry of hyaloid cavity tissue section as an assessment of blood vessel endothelial cells present . Data of individual animals are displayed as circles with means and standard error of the mean and were analysed by unpaired , two - tailed Student ' s t - test .

O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Densitometry of hyaloid cavity tissue section as an assessment of blood vessel endothelial cells present. Data of individual animals are displayed as circles with means and standard error of the mean and were analysed by unpaired, two-tailed Student's t-test.