Representative IF staining for Mash1 , Tuj1 , Cas3 and Ki67 in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + REST DKD mice . Scale bars : 20 μm . F - I Quantification of the number of Mash1 + ( F ) , Tuj1 + ( G ) , Cas3 + ( H ) and Ki67 + ( I ) cells in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + REST DKD mice ( n = 4 mice each group ; data represent the mean ± SEM ; Mash1 : F = 35 . 53 , Tuj1 : F = 62 . 28 , Cas3 : F = 115 . 7 , Ki67 : F = 77 . 43 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_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

Representative IF staining for Mash1, Tuj1, Cas3 and Ki67 in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice. Scale bars: 20 μm. F-I Quantification of the number of Mash1+(F), Tuj1+(G), Cas3+(H) and Ki67+(I) cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice (n=4 mice each group; data represent the mean±SEM; Mash1: F=35.53, Tuj1: F=62.28, Cas3: F=115.7, Ki67: F=77.43, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).


The rate of glucose uptake ( a , e ) , lactate released after 4 h of incubation ( b , f ) , glycolytic flux ( c , g ) and pentose - phosphate flux ( d , h ) were assessed in MEF and mouse cortical neurons in primary culture , as indicated , obtained from either WT or Pink1 KO ( Pink1 − / − ) mice .

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The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.


Blood concentrations of glucose ( i ) and lactate ( j ) , and the rate of 14C - lactate appearance in the blood from acutely injected [ U - 14C ] glucose in the tail vein ( k ) of WT and Pink1 KO mice were analysed . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO miceMEF ( a ) ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT .

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Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO miceMEF (a) ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null mice MEF ( b ) ß - Actin was used as loading control .

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Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null mice MEF (b) ß-Actin was used as loading control.


Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO neurons ( c ) . ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT .

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Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null neurons ( d ) ; ß - Actin was used as loading control .

Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null neurons (d); ß-Actin was used as loading control.


( e ) Western blot against PINK1 , GLUT1 , GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples ; α - Tubulin was used as loading control . Only representative western blots are shown per condition ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . The mean ± s . e . m . values of the mRNA data were calculated from the fold change of each ß - Actin - normalized transcript abundance in the Pink1 KO samples versus that in the WT . Thus , in all cases , the WT values were considered = 1 . 00 . * P0 . 05 versus the corresponding WT ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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(e) Western blot against PINK1, GLUT1, GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples; α-Tubulin was used as loading control. Only representative western blots are shown per condition; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-Actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT. Thus, in all cases, the WT values were considered=1.00. *P0.05 versus the corresponding WT (Student's t-test; n=3-4 independent experiments).


( a ) RT - qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α , or siControl ( 100 nM ) , after 72 h .

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(a) RT-qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α, or siControl (100 nM), after 72 h.


( b ) siHif1α effectively knockdowns expressed Hif1α cDNA WT , but not a Hif1α cDNA silent mutant form refractory to siHif1α ( Hif1α cDNA REFR . ) in mice MEF .

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(b) siHif1α effectively knockdowns expressed Hif1α cDNA WT, but not a Hif1α cDNA silent mutant form refractory to siHif1α (Hif1α cDNA REFR.) in mice MEF.


( c ) RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α . ß - Actin mRNA was used for normalization , and all data are expressed as the change relative to WT - siControl .

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(c) RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α. ß-Actin mRNA was used for normalization, and all data are expressed as the change relative to WT-siControl.


( d ) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α ; ß - actin was used as loading control . As shown , siHif1α partially prevented the increase in GLUT1 , HK2 and GAPDH protein abundances .

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(d) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α; ß-actin was used as loading control. As shown, siHif1α partially prevented the increase in GLUT1, HK2 and GAPDH protein abundances.


The increased rate of lactate released after 4 h of incubation ( e ) and glycolytic flux ( f ) observed in the Pink1 KO MEF was prevented by siHif1α .

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The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α.


siHif1α was also effective at preventing the endogenous increased HIF1α protein ( g ) , as well as the increased glycolytic flux ( h ) observed in Pink1 KO primary neurons . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information .

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siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information.


( a ) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase ( CS ) , and ( b ) mitochondrial inner membrane potential ( Δψm ) in WT and Pink1 KO mice MEF .

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(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF.


( c ) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α ( or its siControl ) ; ß - actin was used as loading control .

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(c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control.


d ) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl .

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d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl.


Rate of whole - cell H2O2 ( e ) and mitochondrial O2 · − ( f ) detection in WT and Pink1 KO

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Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF


( g ) Detection of mitochondrial O2 · − using the mitochondrial - specific probe MitoSox in MEF incubated in the absence ( − ) or in the presence ( + ) of the plasma membrane permeable GSH - EE , or by expressing mitochondrial - tagged forms of glutamate - cysteine ligase , ( + mitoGCL ; empty vector , pIRES2 - EGFP , denoted as − ) or catalase ( + mitoCAT ; empty vector denoted as − ) , shows effective mitochondrial ROS removal .

O O O O O O B-MEASURED_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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O

(g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal.


( h ) Western blot against HIF1α in Pink1 KO MEF incubated with ( or without ) GSH - EE , or transfected with empty vector , mitoGCL or mitoCAT , showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS ; α - TUBULIN was used as loading control . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


( a ) Proportion of BrdU incorporation into DNA and cell cycle phases , and ( b ) cell growth in WT and Pink1 KO MEF after 72 h in the absence ( denoted as - 2DG ) or in the presence ( + 2DG ) of the glucose - metabolizing inhibitor , 2 - deoxyglucose ( 2DG , 1 mM ) .

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(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).


( c ) Proportions of BrdU incorporation into DNA and cell cycle phases , and ( d ) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α ( + siHif1α ) or siControl ( denoted as - siHif1α ) .

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(c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α).


( e ) Ki67 , Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain , and the corresponding quantification . * P0 . 05 ; # P0 . 05 versus all other conditions ( analysis of variance followed by Bonferroni test ; n = 3 - 4 independent experiments ) . All data are expressed as mean ± s . e . m . Scale bar , 100 μm .

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(e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3-4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.


a ) Rate of mitochondrial O2 · - detection in WT and Pink1 KO mice primary neurons .

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a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons.


( b ) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons .

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(b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons.


( c ) Proportion of apoptosis in WT and Pink1 KO neurons , as assessed by the proportion of annexin V - positive / 7 - AAD - negative cells , determined by flow cytometry . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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(c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


( A , B ) Airyscan confocal analysis of PC1 ( 568 , red ) co - localization with GFP - LC3 ( green ) in ( A ) MEF ( B ) Saos2 . Scale bars = 10 µm . ( C ) Airyscan confocal analysis of PC2 ( 647 , red ) co - localization with LC3 ( 488 , green ) in RCS cells . Scale bars = 10 µm . ( A - C ) The insets show higher magnification ( A = x4 . 68 ; B = x6 . 76 ; C = x7 . 33 ) and single colour channels of the boxed area . ( D ) Quantification of GFP ( A , B ) or LC3 ( C ) vesicles positive for PC1 or PC2 , expressed as % of total LC3 ( mean + / - SEM ) , n = 18 cells ( MEFs and Saos2 ) ; n = 12 ( RCS ) from 3 independent experiments .

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(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm. (C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area. (D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments.


( E - G ) Scanning confocal microscopy analysis of MEFs , Saos2 and RCS cells treated with BafA1 , immunolabeled for PC1 or PC2 and LAMP1 . Nuclei were stained with Hoechst . ( E , F ) Scale bars = 10 µm , ( G ) Scale bars = 5 µm . The insets show higher magnification ( E = x4 . 99 ; F = x6 . 49 ; G = x2 . 01 ) and single colour channels of the boxed area .

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(E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.


( H ) Transmission EM analysis in Saos2 cells , treated with BafA1 , showing in detail a lysosome which contains immunolabeled PC1 ( with nanogold particles ) , as indicated by arrows .

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(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.


( I ) Scanning confocal microscopy analysis of Saos2 WT and CRISPR - Cas9 IDUA Saos2 at steady state , immunolabeled for PC1 and LAMP1 . Nuclei were stained with Hoechst . Scale bar = 10 µm . The insets show higher magnification ( left = x3 . 09 ; right = x3 . 12 ) and single colour channels of the boxed area . Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell ( mean + / - SEM ) . n = 31 WT cells , n = 33 CRISPR cells counted ; 3 independent experiments . Student ' s unpaired , two - tailed T - Test * * * P < 0 . 0001 .

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(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.


( J ) WT and CRISPR IDUA Saos2 lysed and analysed by western blot . Data are representative of 3 independent experiments .

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(J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.


U2OS expressing ( A ) GFP - LC3 mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification ( A = x3 . 93 from the boxed region are shown on the right . Scale bar = 10 µm .

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U2OS expressing (A) GFP-LC3 mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93 from the boxed region are shown on the right. Scale bar = 10 µm.


U2OS expressing ( B ) GFP - 2 - FYVE ( green ) , mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification B = x3 . 42 from the boxed region are shown on the right . Scale bar = 10 µm .

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U2OS expressing (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification B = x3.42 from the boxed region are shown on the right. Scale bar = 10 µm.


( C ) Airyscan analysis of Saos2 cells expressing GFP - LC3 ( green ) and immunolabeled for PC1 ( 405 , blue ) and CANX ( 647 , red ) . The insets show higher magnification ( x5 . 26 ) and single colour channels of the boxed area . Scale bar = 10 µm .

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(C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm.


( D ) Correlative Light Electron Microscopy ( CLEM ) and Electron Tomography of Saos2 cells transfected with GFP - LC3 ( green ) and labelled for PC1 ( 568 , red and nanogold particles ) and CANX ( 647 , blue ) . Cells were first imaged by confocal microscopy ( top left panel ) and then the same region was retraced in EM ( upper middle panel ) and overlay is shown ( upper right panel ) . Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules .

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(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.


( E ) Single tomography slice ( left panel , taken from boxed are in D at a magnification of x2 . 84 ) , overlay with immunofluorescence ( IF ) ( central panel ) and IF 3D rendering of AV ( green ) and the CANX positive vesicle containing gold particles of labelled collagen ( blue and white respectively ) inside an AV ( right panel ) .

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(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).


( A ) . Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of total number of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h . n = 20 cells per condition ; three independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * * P < 0 . 0001 .

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(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.


( B ) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1 , fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . Scale bar = 10 µm . Insets show magnification of the boxed area . Bar graph ( on the left ) shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 12 , 10 , 12 , 10 , 7 , 10 cells per genotype respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

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(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.


( A ) WT and CRISPR - Cas9 Fam134b knockout MEFs were treated as indicated , lysed and analysed by western blot with the indicated antibodies . Western blots are representative of 4 independent experiments .

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(A) WT and CRISPR-Cas9 Fam134b knockout MEFs were treated as indicated, lysed and analysed by western blot with the indicated antibodies. Western blots are representative of 4 independent experiments.


( B ) WT and CRISPR - Cas9 Fam134b MEFs were immunolabeled for PC1 ( 568 , red ) , nuclei stained with Hoechst ( blue ) and analysed by scanning confocal microscopy . Scale bar = 10 µm .

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(B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm.


( C ) CRISPR FAM134B MEF mock , wild type FAM134B - HA or FAM134Blir - HA transfected were immunolabeled for PC1 ( 568 , red ) , LAMP1 ( 488 , green ) and HA ( 647 , violet ) and analysed by scanning confocal microscopy . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , quantification of n = 10 cells per treatment ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test was performed . ns ≥ 0 . 05 , * * * P < 0 . 0001 .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_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 B-MEASURED_VAR O O O B-MEASURED_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 O O O O

(C) CRISPR FAM134B MEF mock, wild type FAM134B-HA or FAM134Blir-HA transfected were immunolabeled for PC1 (568, red), LAMP1 (488, green) and HA (647, violet) and analysed by scanning confocal microscopy. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), quantification of n=10 cells per treatment; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed. ns ≥0.05, *** P<0.0001.


( A ) Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes , treated with 100 nM BafA1 for 9h . n = 18 cells / treatment ; 3 independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * P < 0 . 005 , * * * P < 0 . 0001 .

O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_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 O O O O O 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) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001.


( B ) WT and Canx - / - MEFs were untreated or treated with BafA1 ( 10 μM ) for 6 h , lysed and analysed by western blot with indicated antibodies . Filamin and β - actin were used as loading control . Dashed line indicates that unnecessary lanes were removed . Western blot is representative of 3 independent experiments .

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(B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments.


( C ) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . CST was added where indicated . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 8 , 8 , 12 , 8 , 8 , 8 cells respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_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 O O O O O O O O O O O

(C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.


( A , B ) Volcano plot comparing protein fold changes between WT versus Fam134b - / - ( A ) and WT versus Canx - / - MEFs ( B ) . Significantly regulated proteins are labelled in red ( log2 fold change > 1 , - log10 p > 1 . 3 ) . Red dots with blue ring indicate collagen 1 peptides . Graphs represent statistics from three separate experiments for each genotype .

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(A, B) Volcano plot comparing protein fold changes between WT versus Fam134b-/- (A) and WT versus Canx-/- MEFs (B). Significantly regulated proteins are labelled in red (log2 fold change > 1, -log10 p>1.3). Red dots with blue ring indicate collagen 1 peptides. Graphs represent statistics from three separate experiments for each genotype.


( B ) U2OS cells were transfected with empty vector control ( pcDNA3 ) , FAM134B - HA WT or mutant constructs as indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against CANX , FAM134B 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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(B) U2OS cells were transfected with empty vector control (pcDNA3), FAM134B-HA WT or mutant constructs as indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against CANX, FAM134B 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


U2OS cells were transfected with HALO - PC2 , FAM134B - HA or FAM134Blir - HA constructs , treated with 100 nM BafA1 for 6h and with CST where indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against HALO , CANX , FAM134B , LC3 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


( B ) SEC trace and SDS - PAGE of purified NHE9 * ( residues 8 to and 574 out of 644 ) as depicted by schematic .

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(B) SEC trace and SDS-PAGE of purified NHE9* (residues 8 to and 574 out of 644) as depicted by schematic.


( C ) Activity of NHE9 * co - reconstituted with ATPase into liposomes to mimic the in vivo situation ( schematic ) . Representative ACMA fluorescence traces of liposome reconstituted NHE9 * ( blue ) , NHE9 * double - mutant N243A - D244A ( red ) , and rat fructose transporter GLUT5 ( black ) . ATP - driven H + pumping establishes a ΔpH ( 0 - 3 min ) . H + efflux is initiated by the addition of 40 mM NaCl , and subsequent addition of NH4Cl ( 4 min ) collapses the proton gradient .

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(C) Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red), and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0-3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient.


( D ) Michaelis - Menten kinetics for NHE9 * ( red ) , NHE9 ΔCTD ( green ) , N243A - D244A ( blue ) and rat GLUT5 ( black ) as detected by ACMA dequenching following substrate addition . In all experiments errors bars , s . e . m . ; n = 3 technical repeats . The apparent KM values are an average from n = 3 separate protein reconstitutions .

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(D) Michaelis-Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments errors bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions.


A ) Native mass spectrum of NHE9 * purified in the presence ( top panel ) or the absence of additional brain lipids ( bottom panel ) . In the presence of brain lipids , NHE9 * is detected as exclusively dimeric protein with poorly resolved peaks , indicating the presence of multiple lipid adducts . Without brain lipid addition , NHE9 * appears as sharp peaks , revealing several adducts of approximately 1 kDa each , as well as a minor monomer population .

O O O O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A) Native mass spectrum of NHE9* purified in the presence (top panel) or the absence of additional brain lipids (bottom panel). In the presence of brain lipids, NHE9* is detected as exclusively dimeric protein with poorly resolved peaks, indicating the presence of multiple lipid adducts. Without brain lipid addition, NHE9* appears as sharp peaks, revealing several adducts of approximately 1 kDa each, as well as a minor monomer population.


B ) Thermal stabilization of purified dimeric NHE9 * - GFP by lipids . Normalized mean FSEC peak fluorescence before heating ( open bars ) , and after heating and centrifugation ( grey bars ) in presence of different lipids . Data presented are mean values ± data range of n = 4 experiments ( see Materials and Methods ) .

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B) Thermal stabilization of purified dimeric NHE9*-GFP by lipids. Normalized mean FSEC peak fluorescence before heating (open bars), and after heating and centrifugation (grey bars) in presence of different lipids. Data presented are mean values ± data range of n = 4 experiments (see Materials and Methods).


C ) Thermal shift stabilization of purified dimeric NHE9 * - GFP in the presence of PIP2 ( red ) compared to PIP2 - free ( black ) . Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations .

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C) Thermal shift stabilization of purified dimeric NHE9*-GFP in the presence of PIP2 (red) compared to PIP2-free (black). Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


E ) Thermal shift stabilization of the dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in the absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations . ( F ) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts ; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained .

O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. (F) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained.


E ) Thermal shift stabilization of the dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in the absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations .

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E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations.


Verification of screen hits by mtKeima - based FACS analysis of mitophagy . Upper : representative FACS analysis of mitophagy in HeLa cells expressing sgNTC and sgFBXL4 . Bottom : quantitative analysis of mitophagy levels in HeLa cells expressing the indicated sgRNAs . The percentage of cells with high mitophagy level is indicated in red . Two independent sgRNAs were used for each gene . NTC : non - targeting control .

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Verification of screen hits by mtKeima-based FACS analysis of mitophagy. Upper: representative FACS analysis of mitophagy in HeLa cells expressing sgNTC and sgFBXL4. Bottom: quantitative analysis of mitophagy levels in HeLa cells expressing the indicated sgRNAs. The percentage of cells with high mitophagy level is indicated in red. Two independent sgRNAs were used for each gene. NTC: non-targeting control.


Imaging analysis of mitophagy levels in the indicated HeLa cells . Left : sequencing result of WT and FBXL4 - KO cells and representative mitophagy images , white arrows point to mitolysosomes ( FBXL4 - KO cells were not labeled because of the large numbers of mitolysosomes ) ; middle : percentage of cells with mitophagy ( mitolysosome - positive ) ; right : quantification of mitolysosome / mitochondria area . n : number of cells analyzed ( middle ) ; number of imaging areas ( 20 - 30 cells / area ) analyzed ( right ) .

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Imaging analysis of mitophagy levels in the indicated HeLa cells. Left: sequencing result of WT and FBXL4-KO cells and representative mitophagy images, white arrows point to mitolysosomes (FBXL4-KO cells were not labeled because of the large numbers of mitolysosomes); middle: percentage of cells with mitophagy (mitolysosome-positive); right: quantification of mitolysosome/mitochondria area. n: number of cells analyzed (middle); number of imaging areas (20-30 cells/area) analyzed (right).


FACS analysis of mitophagy levels in the indicated HeLa cells . Left : representative FACS results ; right : quantitative analysis of mitophagy levels .

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FACS analysis of mitophagy levels in the indicated HeLa cells. Left: representative FACS results; right: quantitative analysis of mitophagy levels.


Immunoblot analysis of the indicated HeLa cells . B : BNIP3 ; N : NIX . Two clones were shown for each double and triple knockout cells .

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Immunoblot analysis of the indicated HeLa cells. B: BNIP3; N: NIX. Two clones were shown for each double and triple knockout cells.


Representative live cell imaging ( E ) of mitophagy levels in the indicated HeLa cells . n : number of cells analyzed ( left ) ; number of imaging areas analyzed ( right ) .

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Representative live cell imaging (E) of mitophagy levels in the indicated HeLa cells. n: number of cells analyzed (left); number of imaging areas analyzed (right).


Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion . Mitochondria from HeLa FBXL4 - FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X - 100 buffer . Different mitochondrial preparations were then digested with Protease K or trypsin .

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Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion. Mitochondria from HeLa FBXL4-FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X-100 buffer. Different mitochondrial preparations were then digested with Protease K or trypsin.


Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction . Purified mitochondria from HeLa FBXL4 - FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions . S : supernatant ; P : pellet .

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Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction. Purified mitochondria from HeLa FBXL4-FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions. S: supernatant; P: pellet.


Determination of the submitochondrial localization of FBXL4 ( 1 - 23 ) - RFP by Protease K digestion . Experiments were performed similarly as ( A ) .

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Determination of the submitochondrial localization of FBXL4(1-23)-RFP by Protease K digestion. Experiments were performed similarly as (A).


Immunoprecipitation analysis of the FBXL4 - Skp1 - Cullin1 ( SCF - FBXL4 ) complex . HeLa cells were infected with lentiviruses expressing the indicated genes . ∆ F : the F - box deletion mutant of FBXL4 - FLAG ; 4A : FBXL4 - 4A mutant as shown in ( A ) .

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Immunoprecipitation analysis of the FBXL4-Skp1-Cullin1 (SCF-FBXL4) complex. HeLa cells were infected with lentiviruses expressing the indicated genes. ∆F: the F-box deletion mutant of FBXL4-FLAG; 4A: FBXL4-4A mutant as shown in (A).


Immunoprecipitation analysis of the ubiquitination of BNIP3 . FLAG - BNIP3 ( knockin ) , FBXL4 - KO , HA - ubiquitin ( Ub ) HeLa cells were rescued with WT or ∆ F FBXL4 , and treated with DMSO or MG132 ( 20 μM ) for 8 hours . Immunoprecipitation analysis of the ubiquitination of NIX . FLAG - NIX ( knockin ) , FBXL4 - KO , HA - Ub HeLa cells were treated the same as ( F ) .

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Immunoprecipitation analysis of the ubiquitination of BNIP3. FLAG-BNIP3 (knockin), FBXL4-KO, HA-ubiquitin (Ub) HeLa cells were rescued with WT or ∆F FBXL4, and treated with DMSO or MG132 (20 μM) for 8 hours.   Immunoprecipitation analysis of the ubiquitination of NIX. FLAG-NIX (knockin), FBXL4-KO, HA-Ub HeLa cells were treated the same as (F).


Immunoblot analysis of the indicated HeLa cells . FBXL4 - KO HeLa cells were rescued with vector , FBXL4 - FLAG , FBXL4 ( 4A ) - FLAG or FBXL4 ( ∆ F ) - FLAG .

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Immunoblot analysis of the indicated HeLa cells. FBXL4-KO HeLa cells were rescued with vector, FBXL4-FLAG, FBXL4(4A)-FLAG or FBXL4(∆F)-FLAG.


Immunoblot analysis of the indicated HeLa cells . WT and FBXL4 - KO HeLa cells were rescued with vector , WT or mutant FBXL4 - FLAG .

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Immunoblot analysis of the indicated HeLa cells. WT and FBXL4-KO HeLa cells were rescued with vector, WT or mutant FBXL4-FLAG.


Immunoprecipitation analysis of FBXL4 - substrate interaction . FBXL4 - KO HeLa cells were rescued with vector , FBXL4 ( ∆ F ) - FLAG or mutant FBXL4 ( ∆ F ) - FLAG .

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Immunoprecipitation analysis of FBXL4-substrate interaction. FBXL4-KO HeLa cells were rescued with vector, FBXL4(∆F)-FLAG or mutant FBXL4(∆F)-FLAG.


Immunoprecipitation analysis of the integrity of the SCF - FBXL4 complex . Upper : FBXL4 - KO HeLa cells expressing vector , WT or mutant FBXL4 - FLAG were crosslinked with DSP and subject to anti - FLAG immunoprecipitation ; lower : quantification of the ratio of Cullin1 / FLAG band intensities .

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Immunoprecipitation analysis of the integrity of the SCF-FBXL4 complex. Upper: FBXL4-KO HeLa cells expressing vector, WT or mutant FBXL4-FLAG were crosslinked with DSP and subject to anti-FLAG immunoprecipitation; lower: quantification of the ratio of Cullin1/FLAG band intensities.


Viability of the indicated mice . n : number of mice analyzed . Immunoblot analysis of organs from the indicated mice at P0 of age . Elevated BNIP3 and NIX levels are highlighted by red boxes ; reduced mitochondrial proteins are highlighted by blue boxes . Three mice for each genotype were analyzed . Protein samples from HeLa cells treated with DMSO or Actinomycin D ( ActD ) were used as positive control for PARP cleavage .

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Viability of the indicated mice. n: number of mice analyzed.   Immunoblot analysis of organs from the indicated mice at P0 of age. Elevated BNIP3 and NIX levels are highlighted by red boxes; reduced mitochondrial proteins are highlighted by blue boxes. Three mice for each genotype were analyzed. Protein samples from HeLa cells treated with DMSO or Actinomycin D (ActD) were used as positive control for PARP cleavage.


Live cell confocal imaging of mitophagy level in the indicated mouse liver . Left : representative images . The dashed line indicates cell boundary . Right : quantitative analysis of mitophagy levels in hepatocytes . n : number of cells analyzed . Data are mean ± SD . Statistics : two - tailed unpaired Student ' s t - test ; * * * P < 0 . 001 .

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Live cell confocal imaging of mitophagy level in the indicated mouse liver. Left: representative images. The dashed line indicates cell boundary. Right: quantitative analysis of mitophagy levels in hepatocytes. n: number of cells analyzed. Data are mean ± SD. Statistics: two-tailed unpaired Student's t-test; ***P < 0.001.


Viability of the indicated mice . n : number of mice analyzed .

Viability of the indicated mice. n: number of mice analyzed.


Immunoblot analysis of liver samples from the indicated mice at P0 of age . Left : immunoblot analysis , three mice for each genotype were analyzed ; right : quantification of the ratio of mitochondrial protein / actin band intensities . Data are mean + SD from three biological replicates . Statistics : two - tailed unpaired Student ' s t - test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns , not significant .

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Immunoblot analysis of liver samples from the indicated mice at P0 of age. Left: immunoblot analysis, three mice for each genotype were analyzed; right: quantification of the ratio of mitochondrial protein/actin band intensities. Data are mean + SD from three biological replicates. Statistics: two-tailed unpaired Student's t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.


GPS reporter cells expressing peptide libraries ending with a random ( X ) or Gly ( G ) residue were treated with dominant - negative ( DN ) cullins and analyzed . Data are presented as mean ± standard deviation from nine replicates . GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC - box proteins and analyzed . VHL and FEM1C serve as unrelated BC - box protein controls . Data are presented as mean ± standard deviation from nine replicates .

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GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates.


Comparisons of protein stability among wild - type protein and the mutants indicated on the right .

Comparisons of protein stability among wild-type protein and the mutants indicated on the right.


( A ) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations . The number on each graph denotes the left - tailed p - value of the - 1 residue . CT - inside and CT - outside represent membrane proteins with their C - termini facing the cytosol or extracellular space , respectively .

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(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.


( G ) Confocal images of U2OS cells expressing MTSGLUL - GFP proteins with or without a C - terminal diGly degron . MitoTracker staining shows mitochondria . Scale bar = 20 µm . ( H ) Confocal images of U2OS cells expressing MTSGLUL - GFP - diGly degron fusion proteins co - stained with MitoTracker , and with or without DNCul2 treatment . Scale bar = 20 µm .

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(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.


Stability analysis of indicated proteins in cells treated with shRNAs against various BC - box proteins . Since GFP was tagged at the protein N - terminus and signal peptide and MTSs need to be exposed to be functional , all tested proteins were mislocalized . Protein stability analyses of wild - type ( WT ) and mutant proteins lacking their extreme terminal Gly ( ∆ G ) or having it masked by adding Leu ( + L ) in cells with or without BC - box protein knockdown ( C ) or overexpression ( D ) .

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Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized. Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).


Fractionation and Western blot analysis of wild - type and Flag - tagged PDP2 ( top ) , and wild - type and MTS - deleted ( ∆ MTS ) MRPL28 ( bottom ) . Flag - PDP2 and ∆ MTS MRPL28 were mislocalized . W , C , and M denote whole cell lysates , cytosol fraction and mitochondrial fraction , respectively . Tubulin and VDAC serve as fractionation quality controls .

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Fractionation and Western blot analysis of wild-type and Flag-tagged PDP2 (top), and wild-type and MTS-deleted (∆MTS) MRPL28 (bottom). Flag-PDP2 and ∆MTS MRPL28 were mislocalized. W, C, and M denote whole cell lysates, cytosol fraction and mitochondrial fraction, respectively. Tubulin and VDAC serve as fractionation quality controls.


Protein abundance analysis of HA - tagged wild - type or mutant secretory proteins with their signal peptide deleted ( ∆ SP ) in cells with or without KLHDC3 or APPBP2 expression . The HA epitope was inserted immediately after the signal peptide ( SP ) , which had no impact on protein secretion .

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Protein abundance analysis of HA-tagged wild-type or mutant secretory proteins with their signal peptide deleted (∆SP) in cells with or without KLHDC3 or APPBP2 expression. The HA epitope was inserted immediately after the signal peptide (SP), which had no impact on protein secretion.


Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2 , DNCul2 or IMP - 1088 treatments . IMP - 1088 inhibits NMTs . C and M denote cytosol and mitochondrial fractions , respectively . Tubulin and VDAC serve as fractionation quality controls . The relative abundance of cytosolic MIC19 / tubulin was normalized to that of lane 1 and is indicated under each lane in red . Another replicate of this experiment is shown in Fig . EV3B .

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Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.


Abundance comparison of GFP N - or C - terminal tagged with the wild - type or mutant N - terminal ( aa . 1 - 24 ) or C - terminal ( a . a . 218 - 227 ) motif of MIC19 in cells with indicated treatments . Data from sh # 1 is shown here .

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Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here.


Fractionation analysis of wild - type HA - MIC19 in U2OS cells treated with IMP - 1088 and with or without inhibition of ZYG11B or KLHDC2 . The normalized relative abundance of cytosolic HA - MIC19 / tubulin is indicated under each lane in red .

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Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.


GST pull - down assay using cells expressing GST or GST - tagged wild - type or mutant KLHDC2 and HA - tagged SDE2 , FAU or TUG .

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GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.


Relative stability analysis of GAPDH C - terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays . The - 3 , - 4 or - 5 residue of SARS - CoV NSP proteins was mutated to Ala ( A ) as indicated . The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2 . Data are presented as the mean value of GFP / RFP + / - standard deviation from triplicate experiments .

Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.


D , E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 5 mice / group ) . Areas of MCT2 ( upper panel ) and SMCT1 ( lower panel ) stainings ( D ) are expressed as mean of percentage of total tissue area ± SEM . Significance was defined by Mann - Whitney test . * : p < 0 . 05 , * * p < 0 . 01 . Representative images of MCT2 and SMCT1 stainings ( E ) in tumors from KIC mice treated with βOHB or NaCl . Scale bar : 100 µm .

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D, E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel) stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm.


B - D [ U - 13C ] Glucose ( n = 3 mice ) ( B ) , [ U - 13C ] Glutamine ( n = 5 mice ) ( C ) and [ U - 13C ] Acetate ( n = 6 mice ) ( D ) tracing into βOHB in poorly differentiated PDA explants from KIC mice . Data are expressed as mean ± SEM .

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B-D [U-13C]Glucose (n=3 mice) (B), [U-13C]Glutamine (n=5 mice) (C) and [U-13C]Acetate (n=6 mice) (D) tracing into βOHB in poorly differentiated PDA explants from KIC mice. Data are expressed as mean ± SEM.


G Volcano plot illustrating proteins significantly under - or over - represented in sg - CTRL and sg - HMGCL PANC - 1 spheroids ( n = 3 independent experiments ) . Significance was defined by one - tailed Student ' s t - test . Protein levels with a q - value < 0 . 05 ( horizontal axis ) and a fold change < - 1 . 5 or > + 1 . 5 ( vertical axis ) are considered as significantly down or up - regulated in sg - HMGCL PANC - 1 spheroids .

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G Volcano plot illustrating proteins significantly under- or over-represented in sg-CTRL and sg-HMGCL PANC-1 spheroids (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. Protein levels with a q-value<0.05 (horizontal axis) and a fold change <-1.5 or >+1.5 (vertical axis) are considered as significantly down or up-regulated in sg-HMGCL PANC-1 spheroids.


A , B Quantification of volume with representative images ( A ) and weight ( B ) of sg - CTRL ( n = 15 for A and n = 9 for B ) , sg HMGCL # 2 ( n = 19 for A and n = 8 for B ) , # 3 ( n = 7 for A and B ) pancreatic tumors . Data are expressed as mean of tumor volume or weight ± SEM . Significance was defined by Mann - Whitney test . * : p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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A, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors. Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01, ***p<0.001.


C Extracellular matrix quantification following trichrome staining in sg - CTRL and sg - HMGCL # 2 pancreatic tumors sections ( n = 8 and 9 mice / group respectively , left panel ) . Data are expressed as mean of percentage of total tissue area ± SEM . Significance was defined by Mann - Whitney test . * * : p < 0 . 01 . Representative images of trichrome staining in sg - CTRL and sg - HMGCL pancreatic tumors . Scale bar : 100 µm ( right panel ) .

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C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel).


D , E Quantification of volume ( D ) and weight ( E ) of sg - CTRL pancreatic tumors treated with 0 . 9 % NaCl ( i . p . ) ( n = 4 ) , and pancreatic tumors from two different clones of sg - HMGCL treated with 0 . 9 % NaCl ( i . p . ) or βOHB ( 100mg / kg / bi - weekly , i . p . ) ( n = 10 / group ) . Data are expressed as mean of tumor volume or weight ± SEM . Significance was defined by Mann - Whitney test . ns : not significant , * * p < 0 . 01 .

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D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.


H [ U - 13C ] βOHB tracing into TCA intermediate : citrate in sg - CTRL and sg - HMGCL # 2 and # 3 PANC - 1 cells cultured in indicated glucose concentrations . Data are expressed as mean ± SEM . ( n = 2 independent experiments ) . Significance was defined by one - way ANOVA followed by a Bonferroni ' s multiple comparisons test , only significances between sg - HMGCL # 2 , # 3 PANC - 1 cells and sg - CTRL PANC - 1 cells under the same culture condition are mentioned . * * * p < 0 . 001 .

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H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.


( B ) Box - and - whisker plots of ketone bodies ( KB ) metagene score defined as first component of PCA of genes in primary tumors ( n = 728 ) , in all metastases ( n = 76 ) , and in liver metastases specifically ( n = 35 ) . Box - and - whisker plot were defined with default parameters by median value ( central band at the 50th percentile ) , interquartile ranges ( IQR , box limited by 25th and 75th percentile ) and whisker boundaries defined at 1 . 5x IQR . Significance was defined by Student ' s t - test .

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(B) Box-and-whisker plots of ketone bodies (KB) metagene score defined as first component of PCA of genes in primary tumors (n=728), in all metastases (n=76), and in liver metastases specifically (n=35). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined at 1.5x IQR. Significance was defined by Student's t-test.


D - F Effect of βOHB treatment on liver metastatic incidence , size and status . Representative HPS staining of liver lobes from metastatic mice treated with βOHB ( 100mg / kg / day , i . p . ) or 0 . 9 % NaCl ( i . p . ) ( n = 8 mice / group ) . Metastatic areas are separated from liver by yellow dotted lines . Scale bar : 1mm and 500 µm for insets ( D ) . Number and size of metastasis per lobe and classified in small , medium and large size ( E ) . Pathological status of metastasis from mice treated with βOHB or NaCl ( n = 13 or 5 lobes / group , respectively ) . Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis ( F ) .

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D-F Effect of βOHB treatment on liver metastatic incidence, size and status. Representative HPS staining of liver lobes from metastatic mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=8 mice/group). Metastatic areas are separated from liver by yellow dotted lines. Scale bar: 1mm and 500 µm for insets (D). Number and size of metastasis per lobe and classified in small, medium and large size (E). Pathological status of metastasis from mice treated with βOHB or NaCl (n=13 or 5 lobes/group, respectively). Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis (F).


( a , b ) Isolated acini from wild - type ( WT ) and Trpml1 - / - mice ( ML1 - / - ) were used to determine the frequency of Ca2 + oscillations in response to stimulation with 3 and 10 pM CCK ( a ) and the response to high concentrations of CCK ( b ) . The average frequency from multiple experiments is given in the columns as mean ± s . e . m in ( a ) . The traces and columns in ( b ) show the mean ± s . e . m of 3 experiments each includes 4 - 6 acini composed of 6 - 10 cells .

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(a, b) Isolated acini from wild-type (WT) and Trpml1-/- mice (ML1-/-) were used to determine the frequency of Ca2+ oscillations in response to stimulation with 3 and 10 pM CCK (a) and the response to high concentrations of CCK (b). The average frequency from multiple experiments is given in the columns as mean±s.e.m in (a). The traces and columns in (b) show the mean±s.e.m of 3 experiments each includes 4-6 acini composed of 6-10 cells.


( c - e ) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations . ( c ) shows exocytosis during the first 5 min of stimulation , ( d ) shows the two phases , first rapid and second sustained phase , of exocytosis and ( e ) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation . The results are averages of 3 - 4 experiments with each mouse line . denotes p < 0 . 01 or better and # denotes p < 0 . 05 or better .

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(c-e) pancreaticacini were used to measure exocytosis in response to stimulation with the indicated CCK concentrations. (c) shows exocytosis during the first 5 min of stimulation, (d) shows the two phases, first rapid and second sustained phase, of exocytosis and (e) shows exocytosis at the indicated CCK concentrations measured during 30 min of stimulation. The results are averages of 3-4 experiments with each mouse line. denotes p<0.01 or better and # denotes p<0.05 or better.


Panel ( a ) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution . Granules size was analyzed in at least 10 images from 3 wild - type and 5 Trpml1 - / - mice .

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Panel (a) shows example TEM ultrastructure images obtained from male and female mice pancreas and the columns shows the granules size distribution. Granules size was analyzed in at least 10 images from 3 wild-type and 5 Trpml1-/- mice.


( b ) Fixed acini were co - stained with Amylase ( granules ) and LAMP1 ( lysosomes ) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively .

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(b) Fixed acini were co-stained with Amylase (granules) and LAMP1 (lysosomes) and the fold overlap was calculated from 14 and 11 images obtained from 3 wild-type and 3 Trpml1-/- mice, respectively.


( c ) Live wild - type and Trpml1 - / - acini were labeled with mitotracker ( green ) and lysotracker ( red ) . The panels show the merged green , red and DIC images . The large vesicles are marked by white arrows and shown at higher magnification in the inserts . The overlap between lysosomes and granules was determined by ImageJ ( see methods ) . The results are from 14 wild - type and 30 Trpml1 - / - acini each comprising 3 - 7 cells and obtained from 3 wild - type and 4 Trpml1 - / - mice .

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(c) Live wild-type and Trpml1-/- acini were labeled with mitotracker (green) and lysotracker (red). The panels show the merged green, red and DIC images. The large vesicles are marked by white arrows and shown at higher magnification in the inserts. The overlap between lysosomes and granules was determined by ImageJ (see methods). The results are from 14 wild-type and 30 Trpml1-/- acini each comprising 3-7 cells and obtained from 3 wild-type and 4 Trpml1-/- mice.


( d ) Co - IP of lysosomal LAMP1 and granules VAMP8 and Rab27b . Note the increased lysosomal / granules interaction .

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(d) Co-IP of lysosomal LAMP1 and granules VAMP8 and Rab27b. Note the increased lysosomal/granules interaction.


( e ) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1 - / - acini stimulated with 100 pM CCK . The results are the mean ± s . e . m of 3 experiments with acini obtained from 3 wild - type and 3 Trpml1 - / - mice .

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(e) Enhanced exocytosis of the lysosomal acid phosphatase by Trpml1-/-acini stimulated with 100 pM CCK. The results are the mean±s.e.m of 3 experiments with acini obtained from 3 wild-type and 3 Trpml1-/-mice.


( a , b ) Plasmaamylase ( a ) and pancreaticMPO ( b ) in 3 mice of each line .

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(a, b) Plasmaamylase(a) and pancreaticMPO(b) in 3 mice of each line.


( c ) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H & amp ; amp ; E staining . Shown are examples of images and the average damage in the columns , which show the mean ± s . e . m of 4 experiments with each mouse line .

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(c) Edema was evaluated in pancreas of mice treated with PBS or caerulein by H&amp;E staining. Shown are examples of images and the average damage in the columns, which show the mean±s.e.m of 4 experiments with each mouse line.


( d ) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min . Shown are example images and the averages .

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(d) Intracellular trypsin activity was measured in isolated pancreatic acini before and after stimulation with 10 nM CCK for 30 min. Shown are example images and the averages.


( e ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide . The figure shows example images before stimulation and the averaged fluorescence intensity .

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(e) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained for the lipid ceramide. The figure shows example images before stimulation and the averaged fluorescence intensity.


( f ) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3 . The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ . Results in ( c - f ) are given as the mean ± s . e . m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment .

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(f) Isolated acini before and after stimulation with 10 nM CCK for 30 min were stained with the autophagy marker LC3. The figure shows example images before stimulation and the columns depict the number of autophagosomes as determined by ImageJ. Results in (c-f) are given as the mean±s.e.m of 4 experiments from 4 mice of each line and at least 10 acini in each experiment.


( a ) Isolated acini from wild - type ( black ) and Trpml1 - / - mice ( red ) loaded with Fura2 were used to determine [ Ca2 + ] i in response to stimulation with 100 µM carbachol . The traces are the mean ± s . e . m . of 6 acinar clusters . Similar results were obtained in 3 experiments .

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(a) Isolated acini from wild-type (black) and Trpml1-/- mice (red) loaded with Fura2 were used to determine [Ca2+]i in response to stimulation with 100 µM carbachol. The traces are the mean±s.e.m. of 6 acinar clusters. Similar results were obtained in 3 experiments.


( b , c ) Saliva was collected from wild - type ( black ) and Trpml1 - / - mice ( red ) injected I . P . with 10 mg / kg pilocarpine ( b ) or 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol ( c ) over 20 - 40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol . Each condition used 4 - 5 mice from each like and the results are given as the mean ± s . e . m .

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(b, c) Saliva was collected from wild-type (black) and Trpml1-/- mice (red) injected I.P. with 10 mg/kg pilocarpine (b) or 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol (c) over 20-40 min to stimulate fluid secretion by pilocarpine and amylase secretion by isoproterenol. Each condition used 4-5 mice from each like and the results are given as the mean±s.e.m.


( d ) the saliva collected in ( c ) was used to determine the amount of secreted amylase .

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(d) the saliva collected in (c) was used to determine the amount of secreted amylase.


( e , f ) Isolated acini from the parotid glands of wild - type ( black ) or Trpml1 - / - mice ( red ) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times ( e ) and the indicated isoproterenol concentrations during 30 min stimulation ( f ) . The results are the mean ± s . e . m obtained from two mice of each line .

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(e, f) Isolated acini from the parotid glands of wild-type (black) or Trpml1-/- mice (red) were used to measure amylase secretion in response to 50 nM isoproterenol for the indicated times (e) and the indicated isoproterenol concentrations during 30 min stimulation (f). The results are the mean±s.e.m obtained from two mice of each line.


( a ) shows example TEM images and ( b ) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild - type mice and 14 images obtained from 4 Trpml1 - / - mice .

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(a) shows example TEM images and (b) shows the granules size distribution in parotid glands determined from 10 images obtained from 3 wild-type mice and 14 images obtained from 4 Trpml1-/- mice.


( c ) Co - IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b . Input control is actin .

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(c) Co-IP of the lysosomal LAMP1 and the secretory granules VAMP8 and Rab27b. Input control is actin.


( d ) Acini isolated from the parotid glands of wild - type and Trpml1 - / - mice were incubated with mitotracker ( green ) and lysotracker . The large vesicles in the secretory granules / lysosomal area are marked by white arrows . The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line .

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(d) Acini isolated from the parotid glands of wild-type and Trpml1-/- mice were incubated with mitotracker (green) and lysotracker. The large vesicles in the secretory granules/lysosomal area are marked by white arrows. The average number of enlarged profiles is given in the columns and obtained from 3 acinar preparations of each line.


( e ) Shown is the time course ( left panel ) and averaged 10 min secretion ( columns ) of the lysosomal marker acid phosphatase in the saliva collected from wild - type ( black ) and Trpml1 - / - mice ( red ) stimulated with 1 mg / kg pilocarpine and 0 . 6 mg / kg isoproterenol . The mean ± s . e . m is from 4 mice of each line .

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(e) Shown is the time course (left panel) and averaged 10 min secretion (columns) of the lysosomal marker acid phosphatase in the saliva collected from wild-type (black) and Trpml1-/- mice (red) stimulated with 1 mg/kg pilocarpine and 0.6 mg/kg isoproterenol. The mean±s.e.m is from 4 mice of each line.


( a , b ) example traces of mEPSCs recorded from wild - type ( a ) and Trpml1 - / - ( b ) micebrain slices as detailed in Methods . The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale . ( c ) histogram distribution of the events at each mEPSC . The lower columns show the distribution of the higher amplitude mEPSCs . ( d ) the frequency of mEPSCs in each line . The results in ( c , b ) are the mean ± s . e . m of 8 experiments each obtained from 4 mice of each line .

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(a, b) example traces of mEPSCs recorded from wild-type (a) and Trpml1-/-(b) micebrain slices as detailed in Methods. The lower traces in each panel show the portion of the traces marked by dotted boxes in an expanded time scale. (c) histogram distribution of the events at each mEPSC. The lower columns show the distribution of the higher amplitude mEPSCs. (d) the frequency of mEPSCs in each line. The results in (c, b) are the mean±s.e.m of 8 experiments each obtained from 4 mice of each line.


( e ) the mean ± s . e . m of the Ca2 + signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild - type and Trpml1 - / - slices , respectively .

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(e) the mean±s.e.m of the Ca2+ signal in response to depolarization with 30 mM KCl of 6 and 5 experiments with wild-type and Trpml1-/- slices, respectively.


( a ) example TEM images of synapses in wild - type and Trpml1 - / - micebraincortex . ( b ) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild - type and 3 Trpml1 - / - mice , respectively . ( c ) example TEM images at low magnification of wild - type and Trpml1 - / - micebraincortical slices with the dark PSD profiles marked by white arrows . ( d ) average number of PSD / µm2 analyzed in at least 10 images obtained from 3 mice of each line .

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(a) example TEM images of synapses in wild-type and Trpml1-/-micebraincortex. (b) the distribution of synaptic vesicle size determined in 10 and 14 images obtained from 3 wild-type and 3 Trpml1-/-mice, respectively. (c) example TEM images at low magnification of wild-type and Trpml1-/-micebraincortical slices with the dark PSD profiles marked by white arrows. (d) average number of PSD/µm2 analyzed in at least 10 images obtained from 3 mice of each line.


( e ) Glutamate levels and secretion by cultured cerebral cortical neurons . Total glutamate measured in non - stimulated wild - type ( black ) and Trpml1 - / - ( green ) neurons and neurons stimulated by depolarization with 30 mM KCl ( blue and red ) .

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(e) Glutamate levels and secretion by cultured cerebral cortical neurons. Total glutamate measured in non-stimulated wild-type (black) and Trpml1-/- (green) neurons and neurons stimulated by depolarization with 30 mM KCl (blue and red).


( f ) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons . Total glutamate measured in non - stimulated Trpml1 - / - neurons ( green ) and Trpml1 - / - neurons transfected with TRPML1 ( black ) and untransfected ( red ) and TRPML1 - transfected ( clue ) Trpml1 - / - neurons stimulated by depolarization with 30 mM KCl .

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(f) Rescue of glutamate levels and secretion by expression of TRPML1 in cultured cerebral cortical neurons. Total glutamate measured in non-stimulated Trpml1-/- neurons (green) and Trpml1-/- neurons transfected with TRPML1 (black) and untransfected (red) and TRPML1-transfected (clue) Trpml1-/- neurons stimulated by depolarization with 30 mM KCl.


( g ) averaged Co - IP of SNAP25 and VAMP2 in three brain regions of wild - type and Trpml1 - / - mice . The images of all blots are given in Extended view Figure E4 .

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(g) averaged Co-IP of SNAP25 and VAMP2 in three brain regions of wild-type and Trpml1-/- mice. The images of all blots are given in Extended view Figure E4.


293T cells were transfected with a plasmid encoding FLAG - Stx17 wild type ( WT ) or the indicated constructs . At 24 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - FLAG M2 beads , and analyzed by IB using antibodies against PGAM5 and FLAG . Five percent of lysates was analyzed as input .

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293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.


HeLa cells stably expressing FLAG - Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with WT ( paired Student ' s t - test ) .

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HeLa cells stably expressing FLAG-Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with WT (paired Student's t-test).