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

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

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(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 FLAG - Orm2 - 3D . control expresses untagged Orm2 . Input elutions ( with FLAG peptides ) were analyzed by SDS - PAGE Western blot with indicated antibodies .

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(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 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 endoplasmic reticulum ( ER ) . Scale bars = 5 µm .

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(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 Western blot with indicated antibodies of input elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from expressing FLAG - Orm2 - K25 , 33R , FLAG - Orm2 - K25 , 33R - 3A or FLAG - Orm2 - K25 , 33R - 3D . control expresses untagged Orm2 .

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

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

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

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

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

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

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

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

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

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

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

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(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 were treated with or without valinomycin for 40 hr . Total lysates were analyzed by immunoblotting .

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

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(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 − / − , TBC1D15 − / − stably expressing YFP - LC3 were grown in starvation media . Z - stacks of confocal are shown . Scale bars , 20 μm . ( E ) number of YFP - LC3 dots in under growth or starvation conditions was quantified . error bars represent ± SD from three independent replicates . Over 50 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 lysates from grown in normal or starvation media for 6 hr were subjected to immunoblotting . LC3 - I LC3 - II denote 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 − / − those stably expressing HA - TBC1D15 WT HA - TBC1D15 ( Δ221 - 250 ) , FIS1 − / − stably expressing HA - TBC1D15 WT were subjected to immunostaining with anti - TOMM20 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 . were subjected to immunostaining with anti - HA 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 . were subjected to immunostaining with anti - Fis1 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 . extracts were subjected to pull down assays with GFP - Trap . 5 % input bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) 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 . extracts were subjected to pull down assays with GFP - Trap . 5 % input bound fractions were analyzed by immunoblotting with anti - GFP 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 YFP - TBC1D15 WT or YFP - TBC1D17 WT were transiently overexpressed ( OE ) in HeLa . were subjected to immunostaining with anti - HA anti - Fis1 antibodies . of HA Fis1 were merged in 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 ) indicated transiently expressing YFP - GABARAPL1 ( green ) 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 was subjected to binding assays with GST - fused ( GABA , L1 , L2 represent GABARAP , GABARAPL1 , GABARAPL2 , respectively ) . 5 % input bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) . Coomassie brilliant blue ( CBB ) shows GST - fusion in bound fractions ( lower panel ) . ( C ) Binding 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 ) CBB ( 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 ) extracts from HEK293 overexpressed HA - TBC1D15 YFP , YFP - GABARAPL1 ( YFP - WT ) , or its Y49AL50A mutant ( YFP - YL ) were subjected to pull down assays with GFP - Trap . 5 % input bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) 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 ) indicated YFP - tagged TBC1D15 full - , truncated , or point - mutant or YFP - Fis1 overexpressed in HEK293 were subjected to binding assays with recombinant GST - GABARAPL1 . 5 % input bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) CBB ( 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 mCherry - Parkin stably expressing TBC1D15 − / − in presence of HA - tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr . were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( K ) YFP - LC3 of in ( J ) was quantified . error bars represent ± SD from three independent replicates . Over 50 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 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 ) indicated stably expressing YFP - LC3 ( green ) mCherry - Parkin were treated with control ( NTC ) or Rab7 _ # 5 siRNA . After 3 hr valinomycin treatment , were analyzed by immunofluorescence microscopy with anti - TOMM20 antibody ( red ) . Z - stacks of confocal are shown . Magnified are also shown . Scale bars , 10 μm . ( C ) YFP - LC3 morphologies of in ( B ) were quantified . Percentages of harboring diffuse , punctuate or accumulated / tubulated YFP - LC3 are shown . Data error bars were obtained from at least 50 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 ) indicated stably expressing YFP - LC3 ( green ) , mCherry - Parkin , 2HA - Rab7 ( Red ) were treated with or without valinomycin for 3 hr analyzed by immunofluorescence microscopy with anti - HA antibody . Magnified 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 mCherry - Parkin stably expressing TBC1D15 − / − in presence of HA - tagged TBC1D15 WT or D397A mutant were treated with valinomycin for 3 hr . were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( F ) YFP - LC3 of in ( E ) was quantified . error bars represent ± SD from three independent replicates . Over 50 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 4 ) on lipidation of LC3B ( LC3B - II ) were examined by western blotting in absence or presence of a pan - caspase inhibitor Z - VAD ( lanes 3 4 ) compared to vehicle - treated controls ( lanes 1 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 - autophagosomal vesicles ( black arrows ) in absence ( control ) or presence of C18PC was visualized by ( left right , respectively ) . Higher magnification of visualization is shown in lower panels . Scale bars , 10 μm ( top ) 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 in absence or presence of C18PC were determined compared to effects of transfection with Scr siRNAs ( controls ) using Trypan blue exclusion .

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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 regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild - type ( ATG5 + / + ) ATG5 − / − knockout mice with C18PC . Control 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 were determined in MEFs isolated from Bax − / − Bak − / − or Casp3 − / − Casp7 − / − mice 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 absence or presence of C18 - Pyr - Cer ( C18PC ) at 2 h in UM - SCC22A ( 22A ) was examined by visualizing colocalization of MTG ( green ) LTR ( red ) using live - imaging confocal microscopy . Scale bars , 10 μm . Enlarged 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 SeaHorse alongside those of C18PC - 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 its catalytically inactive V5 - tagged mutant ( Mut ) , which cannot generate C18 - ceramide , on formation of LC3B - II were determined by western blotting ( + / − tet ) . Successful induction of wild - type 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 - C18 : 1 - ceramides in response to wild - type CerS1 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 C18 - ceramide induction ( + tet ) on GFP LC3B - GFP lipidation were visualized using confocal microscopy 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 ) effects of induction of wild - type CerS1 C18 - ceramide catalytically inactive mutant CerS1 ( + tet ) in regulation of mitochondrial function were assessed by measuring OCR using SeaHorse compared to roles of noninduced controls ( − tet ) .

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(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 absence ( − tet ) or presence ( + tet ) of wild - type CerS1 C18 - ceramide was visualized by colocalization of MTG LTR using confocal microscopy . Scale bars , 10 μm .

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(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 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 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 mitochondria with anti - ceramide anti - Tom20 , respectively , using confocal microscopy .

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

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(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 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 ) .

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(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 C18 - ceramide induction on lipidation of Flag - tagged wild - type , LC3BI35A , LC3BF52A LC3BG120A were examined by western blotting . Noninduced ( − 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 F52A mutations , which perturbed C18 - ceramide binding of LC3B , on targeting of mitochondria by LC3B - containing autophagosomes ( visualized by confocal microscopy using anti - Flag anti - Tom20 ) ( a ) or mitochondrial function ( measurement of OCR ) ( b ) . V , vector .

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

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(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 regulation of mitochondrial function ( a ) or UM - SCC - 22A xenograft - derived growth ( b ) in absence or presence of wild - type CerS1 C18 - ceramide induction ( − / + tet ) were determined using SeaHorse or measurement of volumes in 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 of control ( CTL - 1 , 2 , 3 ) DOA + fibroblasts carrying indicated monoallelic mutations imaged as described in A . Scale bar = 20μm . Passage number between P12 - 15 . Mitochondrial of B . Data represent mean ± SD of two independent experiments , ( 195 - 2496 per ) , 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 of control ( CTL - 1 ) OPA1S545R fibroblasts treated with OPA1 , DNM1L , or non - targeting ( NT ) siRNAs for 72 hours imaged as described in A . Scale bar = 20μm . Passage number between P12 - 14 . Mitochondrial of D . Data represent mean ± SD of three independent experiments , ( 3219 - 5857 per ) , 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 of control ( CTL - 1 ) OPA1S545R 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 of F . Data represent mean ± SD of two independent experiments , ( 879 - 4154 per ) , 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 of control ( CTL - 1 ) OPA1S545R fibroblasts treated with OPA1 , DNM1L , PGS1 , non - targeting ( NT ) siRNAs or indicated combinations for 72 hours . Mitochondria ( anti - TOM40 , green ) nuclei ( DAPI , blue ) . Scale bar = 20μm . Passages number between P10 - 15 . Mitochondrial of ( A ) using control fibroblasts with fragmented ( OPA1 siRNA ) , normal ( non - targeting NT siRNA ) , hypertubulated ( DNM1L siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 905 - 3695 per ) , ( % fragmented ) ; ; * * * * p < 0 . 0001 , ns ; not significant . .

O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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 of wild type ( WT ) Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours . Live imaging of mitochondria ( mitoYFP , green ) nuclei ( NucBlue , blue ) . Scale bar = 10μm . Mitochondrial 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 per ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant .

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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 of WT , Opa1Crispr MEFs complemented with pLenti - Opa1 , Opa1CrisprPgs1Crispr MEFs Pgs1Crispr MEFs complemented with pLenti - Pgs1 by lentiviral delivery . Live imaging of mitochondria ( mitoYFP , green ) nuclei ( NucBlue , blue ) . Scale bar = 10μm . Supervised ML mitochondrial 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 per ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant . .

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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 of live imaging of MEFs of indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m - chlorophenyl hydrazine ( CCCP ) for indicated time points . were captured every hour for 18 hours . Scale bar = 10μm . Supervised ML mitochondrial 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 per ) , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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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 of live imaging of MEFs of indicated genotypes subjected hyperfusion ( SiMH ) with 10μM cycloheximide ( CHX ) for indicated time points . were captured every hour for 9 hours . Mitochondrial 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 per ) , One - way ANOVA .

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

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

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

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

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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 extracted from WT mutant MEFs were separated by SDS - PAGE ( horizontal denotes separate membranes ) , immunoblotted with indicated antibodies 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 .

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(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 ) cGMP ( solid ) ; right : time course of changes in Vm ( green ) , pHi ( red ) ; [ Na + ] i ( orange ) , [ 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 C , proximal view on left ) cross - sectional ( B D , proximal - to - distal view ) tomographic slices of representative intact flagella from wild - type Chlamydomonas reinhardtii ( C . r . ) flagella ( A B ) sea urchin Strongylocentrotus purpuratus ( S . p . ) sperm ( C D ) . Note that in Chlamydomonas , flagellar ( m ) is surrounded by a glycocalyx ( g ) . Other labels : central pair ( c ) 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 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 . ) n = 18 for Chlamydomonas ( C . r . ) )

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

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

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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 ) normal ASW ( blue , 500 nM cGMP ; navy , 9 nM cGMP ) . time constants of recovery after cGMP release ( 9 nM ) were 289 ms ( depolarization ) 303 ms ( hyperpolarization ) . Signals in panels were detected using potentiometric probe FluoVolt ; dashed magenta lines indicate timing of photolysis flash .

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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 dependence of recovery - time constant on amount of released cGMP . Inset : decay time was constant for low stimulation 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 ) . energy was adjusted such that Vm response amplitudes became similar ; for 8 - Br - cGMP , about 5 - times more energy was required . 10 ms .

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

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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 recorded with FluoForte . UV flash released 28 ( A ) 215 B ) cGMP molecules . 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 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 . D , Average of pHi signals recorded from n = 25 . 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 free cGMP . We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset : expanded scale .

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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 ) weights of E18 . 5 wild - type , TKO QKO foetuses . Data for are presented as circles with mean ± . Data were analysed by one - way ANOVA followed by multiple comparison with Benjamini 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 indicated externally visible developmental defects ( numbers above bars represent number of examined ) . - matched TKO are included for comparison . An example of a QKO E18 . 5 pup presenting with an omphalocele ( asterisk ) , exencephaly ( arrow ) , spina bifida ( arrowhead ) is shown alongside an - 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 indicated defects when examined under a dissection microscope . - matched TKO are included for comparison . Numbers above bars represent number of 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 ) depicting hindlimbs ( HL ) , tail ( T ) , forelimbs ( FL ) , ventral view of palate ( P ) in a wild - type foetus at E18 . 5 of development . E - G ) Corresponding of a representative - matched QKO foetus . Panel G shows a cleft palate ( indicated by arrowheads ) . H , I ) Magnified of panels E F . Arrows indicate excess tissue at anterior posterior edges of hindlimb 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 exhibiting various aortic arch abnormalities great vessel defects ( n = 19 ) .

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


K ) Aortic arch vessels in a wild - type foetus . A corresponding drawing to explain is shown on 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 ) of QKO foetuses accompanying illustration of vessel defects . Absence of right subclavian artery ( L ) 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 stained with H & E . Representative of rostro - occipital level - matched sections from - matched wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( TKO ; n = 4 ) QKO foetuses ( n = 4 ) depicting forebrain , nasal cavity , 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 ) .

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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 are displayed as circles with means standard error of mean . Data were analysed by unpaired , two - Student ' s t - . Related data normalised to mean bodyweight are displayed in Figure EV1 .

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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 eye from E18 . 5 wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( n = 3 ) , Bok - / - Bak - / - Bax - / - Bid - / - quadruple knockout ( n = 3 ) foetuses .

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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 present . Data of are displayed as circles with means standard error of mean were analysed by unpaired , two - Student ' s t - .

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.


( A ) Estimation of BID in TKO MEFs . DKO MEFs re - expressing human BAK , TKO MEFs , two human leukaemia lines RS4 ; 11 MV4 ; 11 , were lysed analysed by Western blot for BID , using a rat monoclonal antibody raised against human BID ( anti - hBID clone 2D1 ) that also recognises mouse BID . Also present are samples of human FL - BID added at equivalent concentrations used in ( B ) ( C ) . On right gel , duplicate samples of DKO TKO permeabilised DKO TKO ( from B C ) showing that endogenous mouse BID is in these . Data shown are representative of 2 independent experiments .

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(A) Estimation of BID protein levels in TKO MEFs. DKO MEFs re-expressing human BAK, TKO MEFs, and two human leukaemia cell lines RS4;11 and MV4;11, were lysed and analysed by Western blot for BID levels, using a rat monoclonal antibody raised against human BID (anti-hBID clone 2D1) that also recognises mouse BID. Also present are samples of the human FL-BID protein added at the equivalent concentrations used in (B) and (C). On the right gel, duplicate samples of DKO and TKO cells and permeabilised DKO and TKO cells (from B and C) showing that endogenous mouse BID is cytosolic in these cells. Data shown are representative of 2 independent experiments.


( B , C ) Partial cytochrome c release by FL - BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID . DKO MEFs re - expressing human BAK or TKO MEFs were permeabilised , then incubated with indicated concentrations of Bim BH3 peptide , cBID or FL - BID , alone or in indicated combinations , at 30oC for 30 min . were then co - stained for cytochrome c TOM20 analysed by flow cytometry . Left panels show median data from replicate experiments ( squares denote experiment 1 , circles denote experiment 2 ) . Right panels show histograms of cytochrome c TOM20 from experiment 2 ( treatments are as listed in left - panels ) .

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(B, C) Partial cytochrome c release by FL-BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID. DKO MEFs re-expressing human BAK or TKO MEFs were permeabilised, and then the cells incubated with the indicated concentrations of Bim BH3 peptide, cBID or FL-BID, alone or in the indicated combinations, at 30oC for 30 min. Cells were then co-stained for cytochrome c and TOM20 and analysed by flow cytometry. Left hand panels show median fluorescence data from replicate experiments (squares denote experiment 1, circles denote experiment 2). Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 (treatments are as listed in the left-hand panels).


B RT - PCR of from mid - temporal autopsy brain sections obtained from NCI , MCI or AD subjects . Representative gel of results is shown . C Quantitation of RT - PCR products of samples analyzed from post - mortem brain tissues ( mean s . e . m . ) . An omnibus significant effect was obtained using one - way of variance ( one - way ANOVA ) with Tukey post - to determine p - value . Error bars are s . e . m .

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B RT-PCR analysis of RNA from mid-temporal autopsy brain sections obtained from NCI, MCI or AD subjects. Representative gel of results is shown.C Quantitation of RT-PCR products of all samples analyzed from post-mortem brain tissues (mean s.e.m.). An omnibus significant effect was obtained using the one-way analysis of variance (one-way ANOVA) with Tukey post-test to determine the p-value. Error bars are s.e.m.


D RT - PCR of from hippocampus of 1 - 2 3 - 7 month old non - transgenic ( WT ) or TgCRND8 ( AD ) mice . E of relative of exon 19 inclusion for TgCRND8 ( AD ) WT mice ( mean s . e . m . ; one - way ANOVA with Tukey post - ) . number of samples analyzed ( n ) is indicated .

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D RT-PCR analysis of RNA from the hippocampus of 1-2 and 3-7 month old non-transgenic (WT) or TgCRND8 (AD) mice.E Quantification of the relative ratio of exon 19 inclusion for TgCRND8 (AD) and WT mice (mean s.e.m.; one-way ANOVA with Tukey post-test). The number of samples analyzed (n) is indicated.


B Top , diagram of ASOs tested below , mapped to their position of complementarity on ApoER2 . Bottom , RT - PCR of isolated from HeLa transfected with indicated ASO at a final of 80 nM . spliced forms are labeled . of inclusion is shown below image calculated as : Inclusion ( % ) = [ exon 19 included / ( exon 19 included + exon 19 skipped ) ] x 100 .

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B Top, diagram of ASOs tested below, mapped to their position of complementarity on ApoER2. Bottom, RT-PCR of RNA isolated from HeLa cells transfected with the indicated ASO at a final concentration of 80 nM. RNA spliced forms are labeled. Quantification of the percentage inclusion is shown below the image and calculated as: Inclusion (%)=[exon 19 included/(exon 19 included+exon 19 skipped)] x 100.


D Top , RT - PCR of ApoER2 exon 19 splicing in HeLa depleted of SRSF1 using an SRSF1 - specific siRNA . Control is a non - specific siRNA . Bottom , Immunoblot of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . E of exon 19 splicing in ( D ) . Error bars show s . e . m . P - value was determined using Student ' s two - t - .

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D Top, RT-PCR analysis of ApoER2 exon 19 splicing in HeLa cells depleted of SRSF1 using an SRSF1-specific siRNA. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.E Quantification of exon 19 splicing in (D). Error bars show s.e.m. P-value was determined using Student's two-tailed t-test.


F Top , RT - PCR of exon 19 splicing in a mouse kidney depleted of SRSF1 by RNAi . Control is a non - specific siRNA . Bottom , Immunoblot of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . G of exon 19 splicing in E . Error bars show s . e . m . P - value was calculated using Student ' s two - t - .

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F Top, RT-PCR analysis of exon 19 splicing in a primary mouse kidney cell line depleted of SRSF1 by RNAi. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.G Quantification of exon 19 splicing in E. Error bars show s.e.m. P-value was calculated using Student's two-tailed t-test.


A Top , ASOs mapped onto mouse ApoER2 exon 19 gene region . Bottom , RT - PCR of isolated from mouse transfected with different ASOs . of exon 19 inclusion is shown below gel image .

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A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image.


B ASO improves exon 19 inclusion in a dose - responsive manner . RT - PCR of from mouse transfected with increasing doses of ASO - 15 . inclusion of exon 19 is shown below gel image . half - maximal effective ( EC50 ) was calculated using GraphPad Prism version 6 . 0 ( GraphPad Software , San Diego , CA ) after fitting data using nonlinear regression with normalized response variable slope .

B ASO improves exon 19 inclusion in a dose-responsive manner. RT-PCR analysis of RNA from mouse cells transfected with increasing doses of ASO-15. The percent inclusion of exon 19 is shown below the gel image. The half-maximal effective concentration (EC50) was calculated using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA) after fitting the data using nonlinear regression with normalized response and variable slope.


D RT - PCR of isolated from hippocampus of mice treated as adults by ICV injection of indicated ASOs . C refers to a non - specific control ASO . E Quantitation of ApoER2 exon 19 inclusion from D ( mean s . e . m . , one - way ANOVA with Bonferroni multiple comparison .

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D RT-PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs. C refers to a non-specific control ASO.E Quantitation of ApoER2 exon 19 inclusion from D (mean s.e.m., one-way ANOVA with Bonferroni multiple comparison test.


A RT - PCR of ApoER2 exon 19 splicing in isolated from hippocampus of 4 month old AD WT mice treated with ASO - 21 or ASO - C at P1 - 2 . B Quantitation of exon 19 splicing . ( mean s . e . m . ; Kruskal - Wallis with Dunn ' s multiple comparison .

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A RT-PCR analysis of ApoER2 exon 19 splicing in RNA isolated from hippocampus of 4 month old AD and WT mice treated with ASO-21 or ASO-C at P1-2.B Quantitation of exon 19 splicing. (mean s.e.m.; Kruskal-Wallis test with Dunn's multiple comparison test.


C Immunoblot for exon 19 containing ApoER2 ( top ) total ApoER2 ( bottom ) . β - actin was used as loading control . D of ApoER2 isoforms shown in C ( mean s . e . m . , student ' s t - ) .

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C Immunoblot for exon 19 containing ApoER2 (top) and total ApoER2 (bottom). β-actin was used as loading control.D Quantification of ApoER2 protein isoforms shown in C (mean s.e.m., student's t-test).


A Input - output curves show hippocampal CA1 field excitatory postsynaptic potential ( fEPSP ) response amplitudes as a function of stimulus delivered to Schaffer collateral afferent . Two - way repeated - measures ANOVA revealed a significant effect of treatment ( F ( 2 , 119 ) = 31 . 62 , p < 0 . 0001 ) with Bonferroni multiple comparisons showing significant differences between ASO - C treated AD - matched ASO - C treated WT ( * p < 0 . 05 ) . Responses from ASO - 21 treated AD mice were not significantly different from ASO - C treated WT or AD mice .

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A Input-output curves show hippocampal CA1 field excitatory postsynaptic potential (fEPSP) response amplitudes as a function of stimulus intensity delivered to Schaffer collateral afferent fibers. Two-way repeated-measures ANOVA revealed a significant effect of treatment (F(2,119) = 31.62, p < 0.0001) with the Bonferroni multiple comparisons test showing significant differences between ASO-C treated AD and age-matched ASO-C treated WT (* p < 0.05). Responses from ASO-21 treated AD mice were not significantly different from ASO-C treated WT or AD mice.


B Representative paired - facilitation ( PPF ) responses from ASO - C treated WT ( top ) , ASO - C treated AD ( middle ) ASO - 21 treated AD ( bottom ) mice are shown to left . Scale bars = 0 . 2 mV by 10 msec apply to traces . Bar graph ( right ) summarizes paired - ratios . PPF was not significantly different in AD compared to WT samples , nor was it affected by ASO - 21 treatment .

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B Representative paired-pulse facilitation (PPF) responses from ASO-C treated WT (top), ASO-C treated AD (middle) and ASO-21 treated AD (bottom) mice are shown to the left. Scale bars = 0.2 mV by 10 msec apply to all traces. Bar graph (right) summarizes the paired-pulse ratios. PPF was not significantly different in AD compared to WT samples, nor was it affected by ASO-21 treatment.


C LTP was unaffected after ASO - 21 treatment similar in AD compared to WT mice . To combine data from multiple slices , individually measured fEPSP rise slopes were normalized to average baseline value taken 0 - 10 minutes before tetanic stimulation ( arrows ) . normalized values are plotted as a function of time for ASO - C treated WT ( gray , left ) , ASO - C treated AD ( red , middle ) ASO - 21 treated AD mice ( blue , right ) . Representative traces taken before 50 - 60 minutes after tetanic stimulation are shown in insets ( scale bars = 0 . 2 mV by 10 msec for traces ) . D Graph summarizes LTP results as of mean fEPSP rise slope measured 50 - 60 minutes after tetanic stimulation divided by that taken 0 - 10 minutes before tetanic stimulation . There was no significant difference among groups . data are shown as mean ± s . e . m are from same hippocampal brain slices ( n = 6 slices from 4 , 3 , 4 WT / ASO - C , AD / ASO - C AD / ASO - 21 , per group , respectively ) .

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C LTP was unaffected after ASO-21 treatment and similar in AD compared to WT mice. To combine data from multiple slices, individually measured fEPSP rise slopes were normalized to the average baseline value taken 0-10 minutes before tetanic stimulation (arrows). The normalized values are plotted as a function of time for ASO-C treated WT (gray, left), ASO-C treated AD (red, middle) and ASO-21 treated AD mice (blue, right). Representative traces taken before and 50-60 minutes after tetanic stimulation are shown in the insets (scale bars = 0.2 mV by 10 msec for all traces).D Graph summarizes LTP results as the ratio of mean fEPSP rise slope measured 50-60 minutes after tetanic stimulation divided by that taken 0-10 minutes before tetanic stimulation. There was no significant difference among groups. All data are shown as mean ± s.e.m and are from the same hippocampal brain slices (n=6 slices from 4, 3, and 4 WT/ASO-C, AD/ASO-C and AD/ASO-21, animals per group, respectively).