( B ) ET of Mic10 - TO . Mic10 expression was induced for 0 h 16 h . Shown are reconstructions of representative noninduced almost completely rescued mitochondria . OM is displayed in clear grey , side of IM that faces matrix is shown in blue . IM side that faces IMS is shown in blue .

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(B) ET of Mic10-TO cells. Mic10 expression was induced for 0 h and 16 h. Shown are reconstructions of representative noninduced and almost completely rescued mitochondria. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


Knockdown ( KD ) of OPA1 in HeLa . WT Mic60 - KO were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 5 d . ( A ) lysates were analyzed by western blotting .

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Knockdown (KD) of OPA1 in HeLa cells. WT and Mic60-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 5 d. (A) Cell lysates were analyzed by western blotting.


( B ) recordings from OPA1 depleted WT Mic60 - KO . Arrows point to a septum .

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(B) TEM recordings from OPA1 depleted WT and Mic60-KO cells. Arrows point to a septum.


( C , D ) of cristae of OPA1 deficient WT ( C ) Mic60 - KO ( D ) . number of CJs was normalized to of OM ( C ) . number of septated mitochondria in Mic60 - KO was estimated ( D ) .

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(C,D) Quantification of the cristae morphology of OPA1 deficient WT (C) and Mic60-KO (D) cells. The number of CJs was normalized to the length of the OM (C). The number of septated mitochondria in Mic60-KO cells was estimated (D).


( E , F ) ET of mitochondria from OPA1 - KD . WT were transfected with a scrambled control ( E ) or siRNA pools against OPA1 ( F ) for 48 h . Reconstructions of representative mitochondria are displayed . OM is displayed in clear grey , side of IM that faces matrix is shown in blue . IM side that faces IMS is shown in blue .

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(E,F) ET of mitochondria from OPA1-KD cells. WT cells were transfected with a scrambled control (E) or siRNA pools against OPA1 (F) for 48 h. Reconstructions of representative mitochondria are displayed. The OM is displayed in clear grey, the side of the IM that faces the matrix is shown in dark blue. The IM side that faces the IMS is shown in light blue.


WT Mic10 - KO were transfected with a scrambled control ( Ctrl . ) or siRNA pools against OPA1 ( OPA1 - KD ) for 72 h . ( A , B ) STED nanoscopy of OPA1 depleted WT ( A ) Mic10 - KO ( B ) . were immunolabeled for Mic60 ATPB .

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WT and Mic10-KO cells were transfected with a scrambled control (Ctrl.) or siRNA pools against OPA1 (OPA1-KD) for 72 h. (A,B) STED nanoscopy of OPA1 depleted WT (A) and Mic10-KO (B) cells. Cells were immunolabeled for Mic60 and ATPB.


( C ) of OPA1 depleted Mic10 - KO . Left : Representative recordings showing CJs marked by arrows in a control a similar view for an OPA1 - KD . CMs are colored in green OM IBM together in magenta . Right : of mitochondria containing CJs or septa junctions . Sections that contained CJ - like structures were analyzed . Of these , number of sections exhibiting septa or cristae structures ( other ) was quantified .

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(C) TEM of OPA1 depleted Mic10-KO cells. Left: Representative TEM recordings showing CJs marked by arrows in a control cell and a similar view for an OPA1-KD cell. The CMs are colored in green and the OM and IBM together in magenta. Right: Quantification of mitochondria containing CJs or septa junctions. Sections that contained CJ-like structures were analyzed. Of these, the number of sections exhibiting septa or cristae structures (other) was quantified.


( D Depletion of ATP5ME in HeLa . were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d . recording ( left ) of number of cristae per mitochondrial section ( right ) .

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(D Depletion of ATP5ME in HeLa cells. Cells were transfected with a scrambled control or siRNA pools against ATP5ME for 4 d. TEM recording (left) and quantification of the number of cristae per mitochondrial section (right).


( E ) depleted of ATP5ME were immunolabeled for Mic60 recorded with STED . arrows point to crossing points of opposite Mic60 distribution bands .

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(E) Cells depleted of ATP5ME were immunolabeled for Mic60 and recorded with STED. The arrows point to crossing points of the opposite Mic60 distribution bands.


( a ) Top : immunoblots for indicated from two independent experiments . Bottom : of changes in total LRRK2 ( n = 6 independent experiments ) . SH - SY5Y were untreated or incubated with 5 μM lactacystin ( Lact ) , 10 mM 3MA or 20 mM NH4Cl 100 μM leupeptin ( NL ) for 9 . 5 h .

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(a) Top: immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of changes in total LRRK2 (n = 6 independent experiments). SH-SY5Y cells were untreated or incubated with 5 μM lactacystin (Lact), 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin (NL) for 9.5 h.


( b ) Top : representative immunoblots for indicated from two independent experiments . Bottom : of LRRK2 relative to that in untreated samples ( n = 3 or 4 independent experiments ) . Brain slices from mouse cortex , midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 ° C .

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(b) Top: representative immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of LRRK2 relative to that in untreated samples (n = 3 or 4 independent experiments). Brain slices from mouse cortex, midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 °C.


( c , d ) Homogenate ( Hom ) lysosomal fractions ( Lys ) isolated from SH - SY5Y ( c ) or indicated mouse brain regions ( d ) , immunoblotted for indicated .

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(c,d) Homogenate (Hom) and lysosomal fractions (Lys) isolated from SH-SY5Y cells (c) or the indicated mouse brain regions (d), immunoblotted for the indicated proteins.


( e ) HEK293 expressing GFP - tagged LRRK2 transduced with lentivirus control ( Ctr ) or carrying shRNA against LAMP - 2A ( L2A ( − ) ) , incubated or not with NL for 12 h immunoblotted for indicated .

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(e) HEK293 cells expressing GFP-tagged LRRK2 and transduced with lentivirus control (Ctr) or carrying shRNA against LAMP-2A (L2A(−)), incubated or not with NL for 12 h and immunoblotted for the indicated proteins.


( f ) Immunoblots for LRRK2 LAMP - 2A of homogenates lysosome - enriched fractions isolated from control fibroblasts ( Ctr ) or fibroblasts stably interfered for LAMP - 2A ( L2A ( − ) ) , maintained in presence or absence of serum . Actin LAMP - 1 ( L - 1 ) are shown as loading controls of homogenate lysosomes , respectively . values are mean + s . e . m . ; * P 0 . 05 , unpaired t - . Full - blots gels in Supplementary Figure 12 .

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

(f) Immunoblots for LRRK2 and LAMP-2A of homogenates and lysosome-enriched fractions isolated from control fibroblasts (Ctr) or fibroblasts stably interfered for LAMP-2A (L2A(−)), maintained in the presence or absence of serum. Actin and LAMP-1 (L-1) are shown as loading controls of homogenate and lysosomes, respectively. All values are mean + s.e.m.; *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.


( b ) Binding , association uptake calculated from of LRRK2 in immunoblots of lysosomes from starved rat livers , untreated or pretreated with protease inhibitors ( PI ) incubated with LRRK2 . Inset : representative immunoblot . LAMP - 2A is shown as lysosomal loading control . Input ( Inp ) , one - fifth of LRRK2 added . n = 7 independent experiments . Uptake : difference between LRRK2 associated with lysosomes with without PI .

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(b) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with LRRK2. Inset: representative immunoblot. LAMP-2A is shown as lysosomal loading control. Input (Inp), one-fifth of LRRK2 added. n = 7 independent experiments. Uptake: difference between LRRK2 associated with lysosomes with and without PI.


( c ) Effect of increasing concentrations of LRRK2 on degradation of a radiolabeled pool of by intact lysosomes . Values are of degradation without LRRK2 ( n = 5 or 6 independent experiments ) .

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(c) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes. Values are percentage of the degradation without LRRK2 (n = 5 or 6 independent experiments).


( d ) Association of GAPDH to rat liver lysosomes in presence of increasing concentrations of LRRK2 . Values are of GAPDH associated with lysosomes in absence of LRRK2 ( n = 4 - 6 independent experiments in duplicate ) . Inset : representative immunoblot . Inp : 1 μg of

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(d) Association of GAPDH to rat liver lysosomes in the presence of increasing concentrations of LRRK2. Values are percentage of the GAPDH associated with lysosomes in the absence of LRRK2 (n = 4-6 independent experiments in duplicate). Inset: representative immunoblot. Inp: 1 μg of GAPDH


( e , f ) Association of LRRK2 RNase A ( e ) or LRRK2 GAPDH ( f ) with PI - treated rat liver lysosomes incubated with increasing concentrations of GAPDH ( e ) or RNase A ( f ) or ovalbumin ( Ov ) . Left : representative immunoblots . Right : association expressed as a of value when incubated with lysosomes alone ( n = 5 or 6 independent experiments ) . Inp : RNase A ( 0 . 4 μg ) , LRRK2 ( 0 . 04 μg ) or GAPDH ( 1 μg ) .

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

(e,f) Association of LRRK2 and RNase A (e) or LRRK2 and GAPDH (f) with PI-treated rat liver lysosomes incubated with increasing concentrations of GAPDH (e) or RNase A (f) or ovalbumin (Ov). Left: representative immunoblots. Right: protein association expressed as a percentage of the value when incubated with lysosomes alone (n = 5 or 6 independent experiments). Inp: RNase A (0.4 μg), LRRK2 (0.04 μg) or GAPDH (1 μg).


( g , h ) Association of LRRK2 to lysosomes incubated with GAPDH ( g ) or RNase A ( h ) at 4 ° C ( n = 3 independent experiments ) .

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(g,h) Association of LRRK2 to lysosomes incubated with GAPDH (g) or RNase A (h) at 4 °C (n = 3 independent experiments).


( i , j ) Immunoblots of brain lysosomes incubated with LRRK2 increasing concentrations of GAPDH ( i ) or RNase A ( j ) . values are mean + s . e . m . ; differences with lysosomes incubated with alone were significant at * P 0 . 05 , unpaired t - . Full - blots gels in Supplementary Figure 12 .

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(i,j) Immunoblots of brain lysosomes incubated with LRRK2 and increasing concentrations of GAPDH (i) or RNase A (j). All values are mean + s.e.m.; differences with lysosomes incubated with the protein alone were significant at *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.


( a ) Top : immunoblot for GFP of tet - on HEK293 expressing GFP - tagged WT or G / S LRRK2 pulsed 24 h with doxycycline , 48 h later ( no longer undergoing de novo synthesis ) , incubated without additions ( − ) or with 20 mM NH4Cl 100 μM leupeptin ( NL ) , 5 μM MG132 ( MG ) or 10 mM 3MA . Bottom : inhibition ( n = 3 independent experiments ) .

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(a) Top: immunoblot for GFP of tet-on HEK293 cells expressing GFP-tagged WT or G/S LRRK2 pulsed 24 h with doxycycline and, 48 h later (no longer undergoing de novo synthesis), incubated without additions (−) or with 20 mM NH4Cl and 100 μM leupeptin (NL), 5 μM MG132 (MG) or 10 mM 3MA. Bottom: percentage inhibition (n = 3 independent experiments).


( a ) Top : immunoblot for GFP of tet - on HEK293 expressing GFP - tagged WT or G / S LRRK2 pulsed 24 h with doxycycline , 48 h later ( no longer undergoing de novo synthesis ) , ( b ) Same as in a , untreated ( Ctr ) or treated with interference for LAMP - 2A ( L2A ( − ) ) , pulsed as in a , sequentially collected immunoblotted . Top left panels : representative immunoblots for GFP . Actin is shown as a loading control . Bottom left panels , , corrected by of knockdown loading control ( n = 3 or 4 independent experiments ) . Top right : immunoblot for LAMP - 2A . Bottom right : of LRRK2 degraded per hour in same samples .

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

(a) Top: immunoblot for GFP of tet-on HEK293 cells expressing GFP-tagged WT or G/S LRRK2 pulsed 24 h with doxycycline and, 48 h later (no longer undergoing de novo synthesis), (b) Same cells as in a, untreated (Ctr) or treated with RNA interference for LAMP-2A (L2A(−)), pulsed as in a, sequentially collected and immunoblotted. Top left panels: representative immunoblots for GFP. Actin is shown as a loading control. Bottom left panels, quantification, corrected by levels of knockdown and loading control (n = 3 or 4 independent experiments). Top right: immunoblot for LAMP-2A. Bottom right: percentage of LRRK2 degraded per hour in the same samples.


( c ) Binding , association uptake calculated from of LRRK2 in immunoblots of lysosomes from starved rat livers , untreated or pretreated with protease inhibitors ( PI ) incubated with WT or G / S LRRK2 . Inset : representative immunoblot ( n = 9 independent experiments ) .

O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O

(c) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with WT or G/S LRRK2. Inset: representative immunoblot (n = 9 independent experiments).


( d ) Association of GAPDH ( 5 μg ) to starved rat liver lysosomes in presence of increasing concentrations of WT or G / S LRRK2 . Inset : representative immunoblot n = 4 - 6 .

O O O O O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O

(d) Association of GAPDH (5 μg) to starved rat liver lysosomes in the presence of increasing concentrations of WT or G/S LRRK2. Inset: representative immunoblot n = 4-6.


( e , f ) Binding of WT G / S LRRK2 to lysosomes in presence of GAPDH ( e ) or RNase A ( f ) . Top : representative immunoblots . Bottom : lysosome - bound LRRK2 expressed as a multiple of amount bound when added alone . Trends of mean values ( n = 2 independent experiments ) ; error bars indicate range .

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(e,f) Binding of WT and G/S LRRK2 to lysosomes in the presence of GAPDH (e) or RNase A (f). Top: representative immunoblots. Bottom: lysosome-bound LRRK2 expressed as a multiple of the amount bound when added alone. Trends of mean values (n = 2 independent experiments); error bars indicate range.


( g ) Immunoblot of rat brain lysosomes ( Lysosom ) incubated with WT or G / S LRRK2 as in c . Inputs , one - tenth of added .

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(g) Immunoblot of rat brain lysosomes (Lysosom) incubated with WT or G/S LRRK2 as in c. Inputs, one-tenth of protein added.


( h , i ) Rat brain lysosomes incubated as in d e , respectively . Left : representative immunoblots . Right : quantifications ( n = 3 or 4 independent experiments ) . values expressed as mean + s . e . m . ; differences with untreated samples ( * ) between WT LRRK2 G / S LRRK2 ( § ) were significant at P 0 . 05 ; n . s . , not significant ; ANOVA Bonferroni . Full - blots gels in Supplementary Figure 12 .

O O O O O B-ORGANISM B-TISSUE B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h,i) Rat brain lysosomes incubated as in d and e, respectively. Left: representative immunoblots. Right: quantifications (n = 3 or 4 independent experiments). All values expressed as mean + s.e.m.; differences with untreated samples (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; n.s., not significant; ANOVA and Bonferroni test. Full-length blots and gels in Supplementary Figure 12.


( a ) Proteolysis in HEK293 stably transfected with an empty plasmid ( Ctr ) or plasmids expressing WT or G / S LRRK2 under control of a tet promoter , treated with doxycycline to activate expression then labeled with [ 3H ] leucine for 48 h . After extensive washing , degradation at indicated times was measured , in maintained either in presence ( + ) or absence ( - ) of serum , as amount of acid - precipitable radioactivity ( small peptides ) released into media . Values are expressed as of radioactivity in ( acid precipitable ) at time 0 ( n = 4 independent experiments , each in triplicate ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proteolysis in HEK293 cells stably transfected with an empty plasmid (Ctr) or plasmids expressing WT or G/S LRRK2 under the control of a tet promoter, treated with doxycycline to activate protein expression and then labeled with [3H]leucine for 48 h. After extensive washing, protein degradation at the indicated times was measured, in cells maintained either in the presence (+) or absence (-) of serum, as the amount of acid-precipitable radioactivity (amino acids and small peptides) released into the media. Values are expressed as percentage of radioactivity in proteins (acid precipitable) at time 0 (n = 4 independent experiments, each in triplicate).


( b ) same , transiently transfected with a KFERQ - PA - mCherry1 reporter for CMA activity . After photoactivation , were maintained for 16 h in presence or absence of serum . Left : representative ; right column shows higher magnification of outlined regions . Arrows denote . Right : average number of per in each condition , measured in > 150 .

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

(b) The same cells, transiently transfected with a KFERQ-PA-mCherry1 fluorescent reporter for CMA activity. After photoactivation, cells were maintained for 16 h in the presence or absence of serum. Left: representative micrographs; right column shows higher magnification images of outlined regions. Arrows denote puncta. Right: average number of puncta per cell in each condition, measured in >150 cells.


( c ) Cultured ventral midbrain dopaminergic neurons from Ctr from WT - LRRK2 G / S - LRRK2 transgenic mice , transduced with same reporter as in b co - stained with antibody to tyrosine hydroxylase ( TH ) . Nuclei are highlighted with DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) in blue . Left : representative . Right : average number of per in > 50 TH - positive . Scale bars , 10 μm . values are mean + s . e . m . ; differences with Ctr ( * ) between WT LRRK2 G / S LRRK2 ( § ) were significant at P 0 . 05 ; unpaired t - in a ANOVA Bonferroni in b , c .

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(c) Cultured ventral midbrain dopaminergic neurons from Ctr and from WT-LRRK2 and G/S-LRRK2 transgenic mice, transduced with the same reporter as in b and co-stained with antibody to tyrosine hydroxylase (TH). Nuclei are highlighted with DAPI (4′,6-diamidino-2-phenylindole) in blue. Left: representative images. Right: average number of puncta per cell in >50 TH-positive cells. Scale bars, 10 μm. All values are mean + s.e.m.; differences with Ctr (*) and between WT LRRK2 and G/S LRRK2 (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c.


( a ) Immunoblots of rat liver lysosomes incubated with WT or G / S LRRK2 alone or with 1 mM GTP / or GAPDH . Duplicate samples are shown . Bottom : of lysosome - bound LRRK2 expressed as a multiple of amount bound without additions ( n = 6 independent experiments ) .

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(a) Immunoblots of rat liver lysosomes incubated with WT or G/S LRRK2 alone or with 1 mM GTP and/or GAPDH. Duplicate samples are shown. Bottom: quantification of lysosome-bound LRRK2 expressed as a multiple of the amount bound without additions (n = 6 independent experiments).


( b ) Effect of kinase inhibitor on WT , G / S , or kinase - dead mutant D / A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH ( 5 μg ) . Right : expressed as a multiple of association without additions ( n = 3 - 5 independent experiments ) .

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(b) Effect of kinase inhibitor on WT, G/S, or the kinase-dead mutant D/A LRRK2 incubated with rat liver lysosomes in presence or absence of GAPDH (5 μg). Right: quantification expressed as a multiple of the association without additions (n = 3-5 independent experiments).


( c , d ) Transient transfection of HEK293 with myc - or GFP - tagged LRRK2 constructs indicated at top left in c . Full , full ; M , KFERQ - like motifs ( M1 - M8 ) . ( c ) Binding to GST - hsc70 . Left , representative immunoblots ; input lanes , one - tenth of input material . NT , non - transfected . Immunoblot for hsc70 is shown as loading control . Top right : of LRRK2 bound to hsc70 . Where indicated , samples were also incubated with RNase A to compete KFERQ - mediated binding of hsc70 ( n = 3 or 4 independent experiments ) . ( d ) Coimmunoprecipitation ( IP ) of hsc70 with anti - myc or anti - GFP in same . Inputs ( Inp ) , one - quarter of starting volume .

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(c,d) Transient transfection of HEK293 cells with the myc- or GFP-tagged LRRK2 constructs indicated at top left in c. Full, full length; M, KFERQ-like motifs (M1-M8). (c) Binding to GST-hsc70. Left, representative immunoblots; input lanes, one-tenth of input material. NT, non-transfected cells. Immunoblot for hsc70 is shown as loading control. Top right: quantification of LRRK2 bound to hsc70. Where indicated, samples were also incubated with RNase A to compete the KFERQ-mediated binding of hsc70 (n = 3 or 4 independent experiments). (d) Coimmunoprecipitation (IP) of hsc70 with anti-myc or anti-GFP in the same cells. Inputs (Inp), one-quarter of starting volume.


( e ) Binding or LRRK2 to rat liver lysosomes treated with indicated concentrations of trypsin . LAMP - 1 is shown to confirm integrity of lysosomes .

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(e) Binding or LRRK2 to rat liver lysosomes treated with the indicated concentrations of trypsin. LAMP-1 is shown to confirm the integrity of the lysosomes.


( f ) Co - IP of LAMP - 2A with anti - LRRK2 in lysosomes incubated with WT or G / S LRRK2 . Inp , one - quarter of starting sample ; IgG , immunoglobulin G . Values : LAMP - 2A recovered in IP ( IP L2A ) corrected for amount of LRRK2 pulled down expressed as a multiple of WT value .

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(f) Co-IP of LAMP-2A with anti-LRRK2 in lysosomes incubated with WT or G/S LRRK2. Inp, one-quarter of starting sample; IgG, immunoglobulin G. Values: LAMP-2A recovered in the IP (IP L2A) corrected for the amount of LRRK2 pulled down and expressed as a multiple of WT value.


( g , h ) Duplicate LAMP - 2A immunoblots of rat liver lysosomes incubated alone ( − ) or with WT or G / S LRRK2 ( g ) or with lysosomes from control ( Ctr ) or from WT - LRRK2 or G / S - LRRK2 transgenic mice ( h ) subjected to blue native electrophoresis . Arrow , 700 - kDa multimeric . values are mean + s . e . m . ; differences compared to none or untreated ( * ) or to WT or full - ( § ) were significant at P 0 . 05 ; unpaired t - in a ANOVA Bonferroni in b , c . Full - blots gels in Supplementary Figure 12 .

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(g,h) Duplicate LAMP-2A immunoblots of rat liver lysosomes incubated alone (−) or with WT or G/S LRRK2 (g) or with lysosomes from control (Ctr) or from WT-LRRK2 or G/S-LRRK2 transgenic mice (h) and subjected to blue native electrophoresis. Arrow, 700-kDa multimeric complex. All values are mean + s.e.m.; differences compared to none or untreated (*) or to WT or full-length protein (§) were significant at P 0.05; unpaired t-test in a and ANOVA and Bonferroni test in b,c. Full-length blots and gels in Supplementary Figure 12.


( a ) Immunofluorescence for LAMP - 2A LAMP - 1 of sections from dorsal motor nuclei of vagus nerves of brains from two different unaffected control ( Ctr ) individuals two individuals with Parkinson ' s disease with G / S LRRK2 mutation . Left : from representative fields . Right : number of per . Black dots , values in ; red lines , mean .

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(a) Immunofluorescence for LAMP-2A and LAMP-1 of sections from the dorsal motor nuclei of the vagus nerves of brains from two different unaffected control (Ctr) individuals and two individuals with Parkinson's disease with the G/S LRRK2 mutation. Left: images from representative fields. Right: number of fluorescent puncta per cell. Black dots, values in individual cells; red lines, mean.


( b ) Immunohistochemistry for LAMP - 2A in same samples .

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(b) Immunohistochemistry for LAMP-2A in the same samples.


( c ) Immunofluorescence for LAMP - 2A ( red ) LRRK2 ( green ) in same samples . Merged from two fluorophore channels are shown ( for colocalization , see Supplementary Fig . 7b ) . Scale bars , 10 μm . Values are mean + s . e . m . ; * P 0 . 05 , ANOVA Bonferroni .

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(c) Immunofluorescence for LAMP-2A (red) and LRRK2 (green) in the same samples. Merged images from the two fluorophore channels are shown (for colocalization quantification, see Supplementary Fig. 7b). Scale bars, 10 μm. Values are mean + s.e.m.; *P 0.05, ANOVA and Bonferroni test.


( a ) Top : LRRK2 immunoblot of rat liver lysosomes incubated with WT , G / S , D / A ( kinase - dead ) , I / T or R / C mutant LRRK2 , alone or in presence of protease inhibitors ( PI ) . Input lanes , one - quarter of input material . Bottom : of binding uptake ( calculated as in Fig . 2b ; n = 4 or 5 independent experiments ) .

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(a) Top: LRRK2 immunoblot of rat liver lysosomes incubated with WT, G/S, D/A (kinase-dead), I/T or R/C mutant LRRK2, alone or in the presence of protease inhibitors (PI). Input lanes, one-quarter of input material. Bottom: quantification of binding and uptake (calculated as in Fig. 2b; n = 4 or 5 independent experiments).


( b ) Left : immunoblots of lysosomes incubated as in a , alone or in presence of increasing concentrations of RNase A ( left ) or GAPDH ( right ) . Right : of LRRK2 bound , as a multiple of binding when incubated with lysosomes alone ( n = 3 independent experiments ) .

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(b) Left: immunoblots of lysosomes incubated as in a, alone or in the presence of increasing concentrations of RNase A (left) or GAPDH (right). Right: quantification of LRRK2 bound, as a multiple of the binding when incubated with lysosomes alone (n = 3 independent experiments).


( c ) Top : coimmunoprecipitation ( co - IP ) of LAMP - 2A ( L2A ) with anti - LRRK2 from lysosomes incubated with LRRK2proteins as in a . Co - immunoprecipitated fractions were immunoblotted for LAMP - 2A LRRK2 . Input : immunoblot for LAMP - 2A before affinity purification . Bottom : amount of LAMP - 2A coimmunoprecipitated , expressed as a multiple of value with WT LRRK2 ( n = 3 or 4 independent experiments ) .

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(c) Top: coimmunoprecipitation (co-IP) of LAMP-2A (L2A) with anti-LRRK2 from lysosomes incubated with LRRK2proteins as in a. Co-immunoprecipitated fractions were immunoblotted for LAMP-2A and LRRK2. Input: immunoblot for LAMP-2A before the affinity purification. Bottom: amount of LAMP-2A coimmunoprecipitated, expressed as a multiple of the value with WT LRRK2 (n = 3 or 4 independent experiments).


( d ) Co - IP of LAMP - 2A with anti - GFP in tet - on HEK293 expressing GFP - LRRK2 pulsed for 24 h with doxycycline . Inputs ( Inp ) , one - quarter of material added .

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(d) Co-IP of LAMP-2A with anti-GFP in tet-on HEK293 cells expressing GFP-LRRK2 proteins and pulsed for 24 h with doxycycline. Inputs (Inp), one-quarter of material added.


( e ) same as in d control transduced with KFERQ - PA - mCherry1 . Left : representative . Nuclei are highlighted with DAPI in blue . Right : after photoactivation 16 h of serum deprivation ( n > 150 ) . values are mean + s . e . m . Differences with WT ( * ) or with indicated mutants ( § ) were significant at P 0 . 05 ; unpaired t - in a - c ANOVA Bonferroni in e . Scale bars , 10 μm . Full - blots gels in Supplementary Figure 12 .

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(e) The same cells as in d and control cells transduced with KFERQ-PA-mCherry1. Left: representative images. Nuclei are highlighted with DAPI in blue. Right: Quantification after photoactivation and 16 h of serum deprivation (n > 150 cells). All values are mean + s.e.m. Differences with WT (*) or with the indicated mutants (§) were significant at P 0.05; unpaired t-test in a-c and ANOVA and Bonferroni test in e. Scale bars, 10 μm. Full-length blots and gels in Supplementary Figure 12.


( a ) α - syn immunoblot of rat liver lysosomes incubated with WT or mutant ( A53T ) α - syn alone ( − ) or with WT LRRK2 or G / S LRRK2 . Inp , input ; Mon , monomers . Right : of α - syn oligomers , expressed as a multiple of amounts in samples without LRRK2 ( n = 4 or 5 independent experiments ) .

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(a) α-syn immunoblot of rat liver lysosomes incubated with WT or mutant (A53T) α-syn alone (−) or with WT LRRK2 or G/S LRRK2. Inp, input; Mon, monomers. Right: quantification of α-syn oligomers, expressed as a multiple of amounts in samples without LRRK2 (n = 4 or 5 independent experiments).


( b - d ) Degradation of radiolabeled ( b ) , binding of GST - α - syn ( c ) of endogenous α - syn ( d ) in intact brain lysosomes ( Lysos ) from non - transgenic ( Ctr ) G / S LRRK2 transgenic mice . Bottom panel in c shows lower exposure to visualize monomers of α - syn . Homog , homogenates ; olig , oligomers ; I , input ( n = 3 independent experiments ) .

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(b-d) Degradation of radiolabeled proteins (b), binding of GST-α-syn (c) and levels of endogenous α-syn (d) in intact brain lysosomes (Lysos) from non-transgenic (Ctr) and G/S LRRK2 transgenic mice. Bottom panel in c shows lower exposure to visualize monomers of α-syn. Homog, homogenates; olig, oligomers; I, input (n = 3 independent experiments).


( e ) Ventral midbraindopaminergic neuronal cultures from control ( Ctr ) or WT - LRRK2 or G / S - LRRK2 transgenic mice untreated or treated with RNAi for LAMP - 2A ( L2A ( − ) ) . ( f ) Colocalization between α - syn total LAMP - 2 from in e ( n > 50 tyrosine hydroxylase ( TH ) - positive neurons ) . Total LAMP - 2 is not reduced in L2A knockdown because LAMP - 2B is upregulated .

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(e) Ventral midbraindopaminergic neuronal cultures from control (Ctr) or WT-LRRK2 or G/S-LRRK2 transgenic mice untreated or treated with RNAi for LAMP-2A (L2A(−)). (f) Colocalization between α-syn and total LAMP-2 from cells in e (n > 50 tyrosine hydroxylase (TH)-positive neurons). Total LAMP-2 is not reduced in L2A knockdown cells because LAMP-2B is upregulated.


( g ) iPSC lines from controls from with G / S LRRK2 - mutation Parkinson ' s disease , differentiated for 30 d immunostained for LAMP - 2A , α - syn TH . Merged channels ( left ) magnified green red channels from boxed region ( right ) . Arrows , colocalization LAMP - 2A α - syn . ( h ) of LAMP - 2A + colocalizing with α - syn in TH + ( n > 50 ) for two control two LRRK2 iPSC lines treated as in g .

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(g) iPSC lines from controls and from patients with G/S LRRK2-mutation Parkinson's disease, differentiated for 30 d and immunostained for LAMP-2A, α-syn and TH. Merged channels (left) and magnified green and red channels from boxed region (right). Arrows, colocalization LAMP-2A and α-syn. (h) Percentage of LAMP-2A+ puncta colocalizing with α-syn in TH+ cells (n > 50) for two control and two LRRK2 iPSC lines treated as in g.


( i , j ) iPSC lines as in g , differentiated for 30 or 75 d untreated or treated with RNAi for LAMP - 2A . ( i ) Immunofluorescence for LAMP - 2A , α - syn GFP in LAMP - 2A - RNAi iPSC lines differentiated for 30 d . Bottom : higher magnifications of boxed areas . ( j ) of TH + showing accumulation of α - syn . n ≥ 2 experiments using two control two LRRK2 iPSC lines ( n > 70 n > 150 TH + in Ctr LRRK2 , respectively ) . Scale bars , 10 μm . values are mean + s . e . m . Differences from WT or control ( * ) or between control or knockdown ( § ) are significant at P 0 . 05 ; unpaired t - in a , h ANOVA Bonferroni in f , j . Full - blots gels in Supplementary Figure 12 .

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(i,j) iPSC lines as in g, differentiated for 30 or 75 d and untreated or treated with RNAi for LAMP-2A. (i) Immunofluorescence for LAMP-2A, α-syn and GFP in LAMP-2A-RNAi iPSC lines differentiated for 30 d. Bottom: higher magnifications of boxed areas. (j) Percentage of TH+ cells showing cytoplasmic accumulation of α-syn. n ≥ 2 experiments using two control and two LRRK2 iPSC lines (n > 70 and n > 150 TH+ cells in Ctr and LRRK2, respectively). Scale bars, 10 μm. All values are mean + s.e.m. Differences from WT or control (*) or between control or knockdown cells (§) are significant at P 0.05; unpaired t-test in a,h and ANOVA and Bonferroni test in f,j. Full-length blots and gels in Supplementary Figure 12.


A . Whole mount in situ hybridization ( WISH ) of evi1at 20 ( left ) 32 ( middle ) hpf . evi1expression is visible in various structures of brain , neuronal structures , posterior pronephric ( ppnd ) branchial arches ( ba ) , as well as in VDA ( ventral dorsal aorta ) region . Additionally , evi1co - localizes with endothelial marker flk1 ( right ) .

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A. Whole mount in situ hybridization (WISH) of evi1at 20 (left) and 32 (middle) hpf. evi1expression is visible in various structures of the brain, neuronal structures, the posterior pronephric duct (ppnd) and the branchial arches (ba), as well as in the VDA (ventral dorsal aorta) region. Additionally, evi1co-localizes with the endothelial marker flk1 (right).


B , C . WISH of runx1 / c - myb ; in HSPCs ( B ) , mpo in neutrophils ( left ) l - plastin ( right ) in monocytes / macrophages ( C ) at 36 hpf in control ( upper ) evi1 MO ( lower ) injected . D - G . WISH of globinin erythrocytes of 6 dpf old ( D ) , of rag - 1in thymus of 5 dpf ( E ; red asterisk ) , of cd41at 52 hpf ( F ) , gata2bat 32 hpf ( G ) for both control ( upper ) evi1 morpholino ( lower ) injected . H . Quantitation of results is shown for each gene , displaying percentages of with normal vs . changed expression in each condition . A Fisher ´ s exact was applied to calculate statistical significance . P < 0 . 001 ( * * * ) .

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B, C. WISH of runx1/c-myb ;in HSPCs (B), mpo in neutrophils (left) and l-plastin (right) in monocytes/macrophages (C) at 36 hpf in control (upper) and evi1 MO (lower) injected embryos.D-G. WISH of globinin erythrocytes of 6 dpf old embryos (D), of rag-1in the thymus of 5 dpf embryos (E; red asterisk), of cd41at 52 hpf (F), and gata2bat 32 hpf (G) for both control (upper) and evi1 morpholino (lower) injected embryos.H. Quantitation of results is shown for each gene, displaying the percentages of embryos with normal vs. changed expression in each condition. A Fisher´s exact test was applied to calculate statistical significance. P < 0.001 (***).


I . WISH of runx1 / c - myb in HSPCs of uninjected control UAS : mEvi1 plasmid injected Tg ( - 1 . 5hsp70l : Gal4 ) with heat - shock induction performed at 14 hpf . J . WISH of runx1 / c - myb in HSPCs of uninjected control UAS : mEvi1 mRNA in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) , leading to endothelial - specific evi1 overexpression . K . Graph displays quantitation of results from I J using same method as above .

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

I. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 plasmid DNA injected Tg(-1.5hsp70l:Gal4) embryos with heat-shock induction performed at 14 hpf.J. WISH of runx1/c-myb in HSPCs of uninjected control and UAS:mEvi1 mRNA in Tg(fli.1:Gal4FF;UAS:RFP) embryos, leading to endothelial-specific evi1 overexpression.K. Graph displays quantitation of results from I and J using the same method as above.


A . WISH of notch1b ( upper ) dll4 ( lower ) in both control ( left ) evi1 MO injected ( right ) .

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

A. WISH of notch1b(upper) and dll4(lower) in both control (left) and evi1 MO injected embryos (right).


B . WISH of notch1b in uninjected ( left ) UAS : mEvi1 plasmid injected ( right ) Tg ( fli . 1 : Gal4FF ; UAS : RFP ) resulting in endothelial - specific evi1 induction .

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

B. WISH of notch1b in uninjected (left) and UAS:mEvi1 plasmid DNA injected (right) Tg(fli.1:Gal4FF;UAS:RFP) embryos resulting in endothelial-specific evi1 induction.


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) from 28 to 42 hpf ( Movie EV 1 ) . Shown are three representative time - points in which transformation from hemogenic endothelial to hematopoietic fate is visible , indicated by arrowheads . For each time - point merged are shown . arrowheads denote double - positive . B . of total numbers of kdrl + cmyb1 + " in view " , dividing respectively emerging HSPCs . n = 10 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney was used to for statistical significance error bars are shown as ± s . d .

O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos from 28 to 42 hpf (Movie EV 1). Shown are three representative time-points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible, indicated by the white arrowheads. For each time-point merged images are shown. White arrowheads denote double-positive cells.B. Cell counts of total numbers of kdrl+cmyb1+ cells "in view", dividing and respectively emerging HSPCs. n=10 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d.


C . Gene expression of endothelial ( kdrl , flt1 , dab2 , cdh5 ) blood specific genes ( c - myb , CD45 , CD41 , runx1 ) in sorted kdrl + c - myb + from 34 hpf Tg ( c - myb : GFP ; kdrl : mKate - CAAX ) . were isolated from 15 - 25 pooled per sample . Three biological experiments were performed with one representative shown . Error bars indicate s . d . of three technical replicates for each representative experiment .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O 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. Gene expression analysis of endothelial (kdrl, flt1, dab2, cdh5) and blood specific genes (c-myb, CD45, CD41, runx1) in sorted kdrl+c-myb+ cells from 34 hpf Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos. Cells were isolated from 15-25 pooled embryos per sample. Three biological experiments were performed with one representative shown. Error bars indicate s.d.
of three technical replicates for each representative experiment.


A . WISH for runx1 / c - mybin 36 hpf evi1 morphant ( left ) evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) ( right ) with global heat - shock ( HS ) induction at 20 hpf . B . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) evi1 morphant transgenic Tg ( 5xUAS - E1b : 6xMYC - notch1a ; cdh5 : gal4ff ) with endothelial - specific NICD induction ( right ) . C . WISH for runx1 / c - myb in 36 hpf evi1 morphant ( left ) evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) ( right ) with global heat - shock induction at 20 hpf . D . WISH for runx1 / c - mybin 32 hpf evi1 MO injected ( left ) injected with both evi1 MO capped gata2 mRNA ( right ) . E . Quantitation of rescue experiments . Shown are percentages of displaying normal or changed runx1 / c - myb expression . A Fisher ´ s exact was applied to calculate statistical significance . ( n . s . , not significant ; p < 0 . 001 ( * * * ) ; p < 0 . 01 ( * * ) ) .

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

A. WISH for runx1/c-mybin 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right) with global heat-shock (HS) induction at 20 hpf.B. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(5xUAS-E1b:6xMYC-notch1a;cdh5:gal4ff) embryos with endothelial-specific NICD induction (right).C. WISH for runx1/c-myb in 36 hpf evi1 morphant (left) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos (right) with global heat-shock induction at 20 hpf.D. WISH for runx1/c-mybin 32 hpf evi1 MO injected embryos (left) and embryos injected with both evi1 MO and capped gata2 mRNA (right).E. Quantitation of all rescue experiments. Shown are the percentages of embryos displaying normal or changed runx1/c-myb expression. A Fisher´s exact test was applied to calculate statistical significance. (n.s., not significant; p < 0.001 (***); p<0.01 (**)).


A . Confocal time - lapse live imaging was performed in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) evi1 morphant ( top , Movie EV 2 ) or in Tg ( c - myb : GFP ; fli . 1 : UAS ; Gal4 : RFP ) crossed to Tg ( 5xUAS - E1b : 6xMYC - notch1a ) prior to MO injection ( bottom , Movie EV 3 ) from 28 to 42 hpf . Shown are three representative time - points , in which transformation from hemogenic endothelial to hematopoietic fate is visible . For each time - point merged are shown . NICD induction controlled by endothelial fli . 1 ( lower panel ) can rescue evi1 morphant phenotype increases number of HSPCs emerging from VDA ( arrows ) . B . of emerging HSPCs in control evi1 morphant fish with or without endothelial NICD induction . At least n = 5 movies from n = 3 biological replicates were analyzed . A non - parametric Mann - Whitney was used to for statistical significance error bars are shown as ± s . d . ( n . s . , not significant , p < 0 . 05 ( * ) ) .

O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) evi1 morphant embryos (top, Movie EV 2) or in Tg(c-myb:GFP;fli.1:UAS;Gal4:RFP) crossed to Tg(5xUAS-E1b:6xMYC-notch1a) embryos prior to MO injection (bottom, Movie EV 3) from 28 to 42 hpf. Shown are three representative time-points, in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible. For each time-point merged images are shown. NICD induction controlled by endothelial fli.1 (lower panel) can rescue the evi1 morphant phenotype and increases the number of HSPCs cells emerging from the VDA (arrows).B. Cell counts of emerging HSPCs in control and evi1 morphant fish with or without endothelial NICD induction. At least n=5 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d. (n.s., not significant, p<0.05 (*)).


A . WISH of dll4 ( upper ) notch1b ( lower ) in control injected ( left ) , evi1 morphants ( middle ) evi1 morphant transgenic Tg ( Hsp70l : vegfaa ; myl7 : eGFP ) at 32 hpf after heat - shock induction at 20 hpf ( right ) .

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

A. WISH of dll4(upper) and notch1b(lower) in control injected embryos (left), evi1 morphants (middle) and evi1 morphant transgenic Tg(Hsp70l:vegfaa;myl7:eGFP) embryos at 32 hpf after heat-shock induction at 20 hpf (right).


B , C . WISH of runx1 / c - myb ( B ) or notch1b ( C ) after treatment with DMSO ( left ) , VEGF receptor inhibitor SU5461 ( middle ) respectively SU5461 after injection of UAS : mEvi1 ( inducing endothelial evi1 expression ) in Tg ( fli . 1 : Gal4FF ; UAS : RFP ) ( right ) .

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

B, C. WISH of runx1/c-myb(B) or notch1b(C) after treatment with DMSO (left), the VEGF receptor inhibitor SU5461 (middle) and respectively SU5461 after injection of UAS:mEvi1 (inducing endothelial evi1 expression) in Tg(fli.1:Gal4FF;UAS:RFP) embryos (right).


A . Expression of runx1 / c - myb in VDA after treatment with DMSO vehicle control ( left ) , PI3K / AKT inhibitor Wortmannin ( WM , middle ) after joined treatment with WM enforced NICD expression via HS at 14 hpf in Tg ( 5xUAS - E1b : 6xMYC - notch1a ; - 1 . 5hsp70l : Gal4 ) ( right ) . Graph displays quantitation of results . Shown are percentages of with normal or decreased runx1 / c - mybexpression for each condition .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

A. Expression of runx1/c-myb in the VDA after treatment with DMSO vehicle control (left), the PI3K/AKT inhibitor Wortmannin (WM, middle) and after joined treatment with WM and enforced NICD expression via HS at 14 hpf in Tg(5xUAS-E1b:6xMYC-notch1a;-1.5hsp70l:Gal4) embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased runx1/c-mybexpression for each condition.


B . Expression of notch1b after treatment with DMSO vehicle control ( left ) or Wortmannin ( WM , middle ) . Graph displays quantitation of results . Shown are percentages of with normal or decreased notch1b expression for each condition .

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

B. Expression of notch1b after treatment with DMSO vehicle control (left) or Wortmannin (WM, middle). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.


C . WISH for runx1 / c - myb in control injected ( upper left ) , evi1 morphants ( upper right ) or respectively evi1 MO with myr - AKT injected with HS at 14 hpf ( lower right ) evi1 MO with UAS - myr - AKT injected transgenic Tg ( fli . 1 : Gal4FFubs3 ; UAS : RFP ) rk . D . Quantitation of results from ( C ) . Shown are percentages of with normal or decreased runx1 / c - myb expression for each condition .

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

C. WISH for runx1/c-myb in control injected embryos (upper left), evi1 morphants (upper right) or respectively evi1 MO with myr-AKT injected embryos with HS at 14 hpf (lower right) and evi1 MO with UAS-myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos.D. Quantitation of results from (C). Shown are the percentages of embryos with normal or decreased runx1/c-myb expression for each condition.


E . WISH for notch1b in evi1 Morpholino injected ( left ) evi1 plus UAS : myr - AKT injected transgenic Tg ( fli . 1 : Gal4FFubs3 ; UAS : RFP ) rk ( right ) . Graph displays quantitation of results . Shown are percentages of with normal or decreased notch1b expression for each condition .

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

E. WISH for notch1b in evi1 Morpholino injected (left) and evi1 plus UAS:myr-AKT injected transgenic Tg(fli.1:Gal4FFubs3; UAS:RFP)rk embryos (right). Graph displays quantitation of results. Shown are the percentages of embryos with normal or decreased notch1b expression for each condition.


( B ) Kaplan - Meier survival curve on n = 17 144DG11 - treated n = 9 vehicle - treated . 144DG11 significantly increased survival ( log - rank p - value < 0 . 000692 ) .

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

(B) Kaplan-Meier survival curve based on n=17 144DG11-treated and n=9 vehicle-treated animals. 144DG11 significantly increased survival (log-rank test p-value<0.000692).


( M ) average duration in movement in an open field as a function of time after treating Gbeys / ys mice with vehicle ( n = 8 ) or 144DG11 ( n = 9 ) at 6 months of ( at onset ) . Data information : Two way ANOVA with repeated measures show that , throughout period , treatment values were not different than vehicle ( p < 0 . 15 ) .

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

(M) average duration in movement in an open field as a function of time after treating Gbeys/ys mice with vehicle (n=8) or 144DG11 (n=9) at 6 months of age (at onset). Data information: Two way ANOVA with repeated measures show that, throughout the period, treatment values were not different than vehicle (p<0.15).


( A ) Left panel , Mice , treated as indicated , were sacrificed indicated tissues were collected stained for PG ( arrows ) with PAS following diastase treatment . Scale bars , 200 µm ( liver , muscle ) , 50 µm ( cortex , heart , peripheral nerve ) . Right panel , PAS was quantified on of 4 sections from each tissue in n = 3 wt , n = 7 Gbeys / ys vehicle - treated , n = 9 144DG11 - treated mice .

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

(A) Left panel, Mice, treated as indicated, were sacrificed and the indicated tissues were collected and stained for PG (arrows) with PAS following diastase treatment. Scale bars, 200 µm (liver, muscle), 50 µm (cortex, heart, peripheral nerve). Right panel, PAS staining was quantified based on analysis of 4 sections from each tissue in n=3 wt, n=7 Gbeys/ys vehicle-treated, and n=9 144DG11-treated mice.


( C ) 144DG11 Pharmacokinetics . Gbeys / ys mice injected with 144DG1 were sacrificed 30 , 60 , 90 , 210 min post injection indicated tissues were removed , as well as 200 uL of serum drawn . Graph shows means ( + / - ) of 144DG11 in different tissues determined by LC - MS / MS Results obtained from n = 3 mice at each time point .

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

(C) 144DG11 Pharmacokinetics. Gbeys/ys mice injected with 144DG1 were sacrificed 30, 60, 90, and 210 min post injection and the indicated tissues were removed, as well as 200 uL of serum drawn. Graph shows means (+/- SEM) of 144DG11 levels in the different tissues determined by LC-MS/MS Results obtained from n = 3 mice at each time point.


A - F Mice were monitored over a 24 hr period . Effective mass was calculated by ANCOVA Data are mean ± from nine month old mice ( n = 11 , wt vehicle - treated , n = 6 Gbeys / ys vehicle - treated , n = 7 Gbeys / ys 144DG11 - treated ) . Vehicle - treated Gbeys / ys mice demonstrate lower respiratory quotient ( in ) ( A ) , total energy expenditure ( TEE ) ( B ) , fat oxidation ( C ) compared to wt controls . 144DG11 treatment increased these parameters ( for fat oxidation only in total time ) . Carbohydrate oxidation ambulatory activity , not significantly affected by diseased state , were increased by 144DG11 even beyond wt control ( D E ) ( note , while 144DG11 increased carbohydrate oxidation in ( D ) , p was only < 0 . 06 ) . 144DG11 also reversed decrease in meal water sip volume observed in Gbeys / ys mice as compared to wt control ( F ) . Data information : * p < 0 . 05 v wt controls , # p < 0 . 05 v Gbeys / ys vehicle treated mice . Statistical differences were determined by two - t - .

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

A-F Mice were monitored over a 24 hr period. Effective mass was calculated by ANCOVA Data are mean±SEM from nine month old mice (n=11, wt vehicle-treated, n=6 Gbeys/ys vehicle-treated, and n=7 Gbeys/ys 144DG11-treated). Vehicle-treated Gbeys/ys mice demonstrate lower respiratory quotient (in the light) (A), total energy expenditure (TEE) (B), and fat oxidation (C) compared to wt controls. 144DG11 treatment increased these parameters (for fat oxidation only in the dark and total time). Carbohydrate oxidation and ambulatory activity, not significantly affected by the diseased state, were increased by 144DG11 even beyond wt control levels (D and E) (note, while 144DG11 increased carbohydrate oxidation in the light (D), p was only <0.06). 144DG11 also reversed the decrease in meal size and water sip volume observed in Gbeys/ys mice as compared to wt control (F). Data information: *p<0.05 v wt controls, #p<0.05 v Gbeys/ys vehicle treated mice. Statistical differences were determined by two-tailed t-tests.


( G ) Blood metabolic panel on n = 3 , 9 . 5 month old mice treated as indicated . Blood glucose was increased blood triglycerides decreased in Gbeys / ys by 144DG11 ( p < 0 . 05 ) .

O O O B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O B-TISSUE B-SMALL_MOLECULE O O B-TISSUE B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O

(G) Blood metabolic panel based on n=3, 9.5 month old mice treated as indicated. Blood glucose was increased and blood triglycerides decreased in Gbeys/ys cells by 144DG11 (p<0.05).


( A ) PAS for total glycogen in skin fibroblasts from different APBD . was quantified by InCell2200 ( see Methods ) . Means ( + / - ) are statistically different from each other ( p < 0 . 0001 , One Way ANOVA ) . n = 3 technical replicates from each indicated at x - axis were used .

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(A) PAS staining for total glycogen in skin fibroblasts from different APBD patients. Staining fluorescence was quantified by InCell2200 (see Methods). Means (+/- SEM) are statistically different from each other (p<0.0001, One Way ANOVA). n=3 technical replicates from each patient indicated at the x-axis were used.


( B ) PAS for total glycogen in APBD87 fibroblasts glucose - starved for 48 h ( left ) , or glucose starved then replenished for last 24 h to induce glycogen burden ( right ) . Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective CY3 filter . Scale bar , 100 µm .

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(B) PAS staining for total glycogen in APBD87 fibroblasts glucose-starved for 48 h (left), or glucose starved and then replenished for the last 24 h to induce glycogen burden (right). Image acquisition performed by Nikon Eclipse Ti2 microscope using a 40x PlanFluor objective and CY3 filter. Scale bar, 100 µm.


( D ) Glycolytic ( red ) mitochondrial ( blue ) ATP production determined by Agilent ' s Seahorse machine real - time ATP rate kit . HC APBD fibroblasts were serum / glucose - starved for 48 h then full medium was replenished for 24 h without ( untreated ) , or with ( chronic ) 50 µM 144DG11 . Acute , 50 µM 144DG11 was added on for 20 min after 24 h of serum / glucose replenishment . Readings were normalized to number as determined by Crystal Violet . Shown are mean values on n = 3 - 6 technical repeats . For experiments ( HC , p < 0 . 01 ; APBD1 , p < 0 . 02 ; APBD2 , p < 0 . 09 ; APBD3 , p < 0 . 11 ; One Way ANOVA with Sidak ' s post - hoc correction for multiple comparisons ) glycolytic ATP production was increased , as compared to untreated control , by acute , but not chronic , supplementation of 144DG11 . Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts ( p < 0 . 06 ) .

O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-DISEASE B-CELL_TYPE O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O B-DISEASE O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-DISEASE B-CELL_TYPE O O O O O O O O

(D) Glycolytic (red) and mitochondrial (blue) ATP production determined by Agilent's Seahorse machine and real-time ATP rate assay kit. HC and APBD patient fibroblasts were serum/glucose-starved for 48 h and then full medium was replenished for 24 h without (untreated), or with (chronic) 50 µM 144DG11. Acute, 50 µM 144DG11 was added on assay for 20 min after 24 h of serum/glucose replenishment. Readings were normalized to cell number as determined by Crystal Violet staining. Shown are mean and SEM values based on n=3-6 technical repeats. For all experiments (HC, p<0.01; APBD1, p<0.02; APBD2, p<0.09; APBD3, p<0.11; One Way ANOVA with Sidak's post-hoc correction for multiple comparisons) glycolytic ATP production was increased, as compared to untreated control, by acute, but not chronic, supplementation of 144DG11. Mitochondrial ATP production was increased only by acute supplementation of 144DG11 to APBD2 fibroblasts (p<0.06).


( C ) thermal shift ( CETSA ) of different targets of 144DG11hetero - assembly ( B ) . Only LAMP1 was significantly protected by 144DG11 from heat - mediated denaturation ( see right shift in its Tm in lower panel ) suggesting its specific interaction with 144DG11 . n = 3 biological replicates . Error bars represent S . D .

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

(C) Cellular thermal shift assay (CETSA) of different targets of the 144DG11hetero-assembly (B). Only LAMP1 was significantly protected by 144DG11 from heat-mediated denaturation (see right shift in its Tm in the lower panel) suggesting its specific interaction with 144DG11. n=3 biological replicates. Error bars represent S.D.


( A ) Left panel , Autophagic flux , determined by extent of lysosomal inhibitors - dependent increase in of lipidated to non - lipidated LC3 ( LC3II / LC3I ) , is increased by 144DG11 ( 50µM , 24h ) . Right panel , 144DG11 - mediated increase in autophagic flux , demonstrated by enhanced degradation of autophagy substrate p62 . Shown are representative immunoblots ( grouped from different gels ) densitometric quantifications + / - s . d . ( for LC3II / LC3I , n = 4 biological replicates , * p < 0 . 0023 ; for p62 , n = 5 biological replicates , * p < 0 . 0099 ; two - t - ) .

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

(A) Left panel, Autophagic flux, determined by the extent of lysosomal inhibitors-dependent increase in the ratio of lipidated to non-lipidated LC3 (LC3II/LC3I), is increased by 144DG11 (50µM, 24h). Right panel, 144DG11-mediated increase in autophagic flux, demonstrated by enhanced degradation of the autophagy substrate p62. Shown are representative immunoblots (grouped from different gels) and densitometric quantifications +/-s.d. (for LC3II/LC3I, n=4 biological replicates, *p<0.0023; for p62, n=5 biological replicates, *p<0.0099; two-tailed t-test).


( B ) Left panel , of liver tissue from vehicle or 144DG11 - treated Gbeys / ys mice . High - magnification ( right , scale bars = 200 nm ) low - magnification ( left , scale bars = 1 µm ) show higher of glycogen / polyglucosan in lysosomes cytosol , respectively . Right panel , ( + / - s . d ) of lysosomal glycogen particles ( n = 3 biological replicates , * p < 0 . 03 , two - t - ) . G , Glycogen / polyglucosan ; L , Lysosomes ; M , Mitochondria .

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(B) Left panel, TEM images of liver tissue from vehicle or 144DG11-treated Gbeys/ys mice. High-magnification (right, scale bars=200 nm) and low-magnification (left, scale bars=1 µm) images show higher levels of glycogen/polyglucosan in lysosomes and cytosol, respectively. Right panel, quantification (+/- s.d) of lysosomal glycogen particles (n=3 biological replicates, *p<0.03, two-tailed t-test). G, Glycogen/polyglucosan; L, Lysosomes; M, Mitochondria.


( C ) 144DG11 reduces LC3 p62 in mouse liver , but not muscle ( n = 3 biological replicates , * p < 0 . 04 for LC3 , * * p < 0 . 0007 for p62 ; two - t - , ) . Scale bars , 10

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(C) 144DG11 reduces LC3 and p62 in mouse liver, but not muscle (n=3 biological replicates, *p<0.04 for LC3, **p<0.0007 for p62; two-tailed t-test, SEM). Scale bars, 10 µm


( E ) LAMP1 - KD 144DG11 treatment cause lysosomal acidification . Upper panel , flow cytometry results showing that 144DG11 slightly increased acidification ( yellow to blue median ( Y / B ) ) in control , GFP - transduced , APBD fibroblasts ( Y / B ( GFP / 144DG11 ) > Y / B ( GFP ) , p < 0 . 12 ) , but significantly acidified LAMP1 - KD , GFP - shLAMP1 - transduced , APBD fibroblasts ( Y / B ( LAMP1 - KD / 144DG11 ) > ( Y / B ( LAMP1 - KD ) , p < 0 . 03 ) . LAMP1 - KD itself led to most significant acidification ( Y / B ( LAMP1 - KD ) > Y / B ( GFP ) , p < 0 . 007 ) . n = 3 , two - t - . Middle panel Lysosensor of corresponding . Yellow correlates with acidification . Lower panel , PAS ( glycogen ) of corresponding . Scale bars , 50 µm .

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

(E) LAMP1-KD and 144DG11 treatment cause lysosomal acidification. Upper panel, flow cytometry results showing that 144DG11 slightly increased acidification (yellow to blue median fluorescence ratio (Y/B)) in control, GFP-transduced, APBD fibroblasts (Y/B(GFP/144DG11)>Y/B(GFP), p<0.12), but significantly acidified LAMP1-KD, GFP-shLAMP1-transduced, APBD fibroblasts (Y/B(LAMP1-KD/144DG11)>(Y/B(LAMP1-KD), p<0.03). LAMP1-KD itself led to the most significant acidification (Y/B (LAMP1-KD)>Y/B(GFP), p<0.007). n=3, two-tailed t-tests. Middle panel Lysosensor staining of the corresponding cells. Yellow fluorescence intensity correlates with acidification. Lower panel, PAS (glycogen) staining of the corresponding cells. Scale bars, 50 µm.


( G ) 144DG11 reduces lysosomal ( LAMP1 positive ) in liver , but not muscle , of Gbeys / ys mice ( n = 3 biological replicates , * p < 0 . 01 , two - t - , ) . Scale bars , 5 µm for liver ( upper left panel ) , 10 µm for muscle ( lower left panel ) . Right panel shows of left panel .

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(G) 144DG11 reduces lysosomal (LAMP1 positive) area in liver, but not muscle, of Gbeys/ys mice (n=3 biological replicates, *p<0.01, two-tailed t-test , SEM).Scale bars, 5 µm for liver (upper left panel), and 10 µm for muscle (lower left panel). Right panel shows quantification of the left panel.


( B ) Morphological characterization of lysosomes on lysotracker of n = 4 HC n = 4 ( biological replicates ) APBD skin fibroblasts starved for 48 h treated or not with 50 uM 144DG11 for 24 h . Values are deviations from untreated HC . 144DG11 has reduced lysotracker mean , integrated ( DXA ) , in both HC APBD fibroblasts ( * * * p < 0 . 0001 , One - Way ANOVA with Sidak ' s multi - comparison post - hoc ) .

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

(B) Morphological characterization of lysosomes based on lysotracker staining of n=4 HC and n=4 (biological replicates) APBD patient skin fibroblasts starved for 48 h and treated or not with 50 uM 144DG11 for 24 h. Values are deviations from untreated HC. 144DG11 has reduced lysotracker mean intensity, integrated intensity (DXA), and area in both HC and APBD fibroblasts (***p<0.0001, One-Way ANOVA with Sidak's multi-comparison post-hoc test).


( C ) Functional ( mitochondrial potential ( TMRE parameters ) ) biomass ( mitotracker ) characterization of mitochondria in HC APBD treated analyzed as in ( B ) . Mean ( * * * , p < 0 . 0001 ) integrated ( * * * , p < 0 . 0006 ) TMRE intensities , mitochondrial biomass ( * * * , p < 0 . 0001 ) , were reduced in APBD v HC . 144DG11 increased TMRE mean ( * * , p < 0 . 009 , * * , p < 0 . 006 ) integrated ( * , p < 0 . 01 , * * , p < 0 . 001 ) intensities in APBD HC , respectively mitotracker in APBD only ( * * * , p < 0 . 0001 ) . Data represent n = 3 biological replicates . Statistical differences analyzed by One - way ANOVA with multiple comparisons .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-DISEASE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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-DISEASE O O O B-SMALL_MOLECULE O B-DISEASE O O O O O O O O O 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) Functional (mitochondrial membrane potential (TMRE parameters)) and biomass (mitotracker) characterization of mitochondria in HC and APBD cells treated and analyzed as in (B). Mean (***, p<0.0001) and integrated (***, p<0.0006) TMRE intensities, and mitochondrial biomass (***, p<0.0001), were reduced in APBD v HC. 144DG11 increased TMRE mean (**, p<0.009, **, p<0.006) and integrated (*, p<0.01, **, p<0.001) intensities in APBD and HC cells, respectively and mitotracker intensity in APBD cells only (***, p<0.0001). Data represent n=3 biological replicates. Statistical differences analyzed by One-way ANOVA with multiple comparisons.


A Agarose gel image illustrating PCR amplicons of ltaS ( LBA0447 ) , slpB , slpX deletions in NCK2187 .

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A Agarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187.


B SDS - PAGE gel of 5 M LiCl ‐ purified S ‐ layer fractions from parental strains , NCK56 NCK1909 , NCK2030 ( LTA + , SlpA + , SlpB − , SlpX − ) , NCK2187 ( LTA − , SlpA + , SlpB − , SlpX − ) .

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

B SDS-PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−).


C B6 mice were orally gavaged with 109 CFU erythromycin ‐ resistant NCK56 or NCK2187 . Fecal pellets were collected daily tested for presence of erythromycin ‐ resistant strains . n = 3 mice / group . Data are representative of five independent experiments are shown as mean ± .

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C B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM.


D ColonicLPL were co ‐ cultured with NCK56 or NCK2187 ( 1 : 1 ) , secreted cytokines were measured in supernatants . Data are shown as mean ± are representative of two experiments performed in triplicate . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

O B-TISSUE O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D ColonicLPL cells were co‐cultured with NCK56 or NCK2187 (1:1), and secreted cytokines were measured in the supernatants. Data are shown as mean ± SEM and are representative of two experiments performed in triplicate. *P 0.05, **P 0.01, ***P 0.001.


A B6 mice were orally gavaged with 109CFU NCK56 ( blue ) or NCK2187 ( green ) on days 0 , 3 , 6 , 9 , or left untreated , immune responses in colon analyzed at day 14 by flow cytometry .

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A B6 mice were orally gavaged with 109CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry.


B , C B6 FoxP3 ‐ GFP mice were treated evaluated as in ( A ) . Regulatory cytokine production in FoxP3 ‐ GFP + ( green ‐ dotted bars ) versus FoxP3 ‐ GFP − ( bars ) was measured by intracellular FACS analyses ( C ) .

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B, C B6 FoxP3‐GFP mice were treated and evaluated as in (A). Regulatory cytokine production in FoxP3‐GFP+ (green‐dotted bars) versus FoxP3‐GFP− (white bars) cells was measured by intracellular staining and FACS analyses (C).


A B6 Rag1 − / − mice were injected with 106CD4 + CD45RBhiT then orally gavaged with NCK56 ( red ) , NCK2187 ( green ) , or SlpA ( blue ) , 1 3 days after transfer , subsequently once a week for four consecutive , or left untreated ( magenta ) . A group of mice was co ‐ transferred with CD4 + CD25 + T as a positive control for protection ( Tregs ; gray ) . Colitis severity was determined in part by loss , diarrhea scores , FOB . See Supplementary Tables S1 , S2 S3 for statistical analyses results .

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A B6 Rag1−/−mice were injected with 106CD4+CD45RBhiT cells and then orally gavaged with NCK56 (red), NCK2187 (green), or SlpA (blue), 1 and 3 days after transfer, and subsequently once a week for four consecutive weeks, or left untreated (magenta). A group of mice was co‐transferred with CD4+CD25+T cells as a positive control for protection (Tregs; gray). Colitis severity was determined in part by weight loss, diarrhea scores, and FOB. See Supplementary Tables S1, S2 and S3 for statistical analyses results.


B , C Colitis scores on histopathology gross of colons of mice treated as in ( A ) were also used as measures of disease . Scale bar = 50 μm .

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B, C Colitis scores based on histopathology and gross morphology of the colons of mice treated as in (A) were also used as measures of disease. Scale bar = 50 μm.


D Circulating of proinflammatory cytokines were measured in sera of mice transferred treated as in ( A ) , or sham adoptive transferred ( bars ) .

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D Circulating levels of proinflammatory cytokines were measured in the sera of the mice transferred and treated as in (A), or sham adoptive transferred (white bars).


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Colonic expression of tight junction ‐ associated genes Cldn3 Ocln , determined by RT - PCR relative to 18S rRNA ( A ) , were used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three experiments are shown as mean ± . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Colonic expression of tight junction‐associated genes Cldn3 and Ocln, determined by RT-PCR relative to 18S rRNA (A), were used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


Colitis was induced in B6 Rag1 − / − mice as described in Fig . A , B Passive transepithelial absorption of FITC ‐ dextran ( B ) , was used as measures of epithelial barrier integrity . Sham adoptive transferred B6 Rag1 − / − mice ( bars ) were used as baseline controls in some cases . n = 5 mice / group . Data represent three experiments are shown as mean ± . * P 0 . 05 , * * P 0 . 01 . Black asterisks compare NCK2187 to PBS ‐ treated adoptively transferred mice , red asterisks to NCK56 ‐ treated mice .

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Colitis was induced in B6 Rag1−/−mice as described in Fig .A, B Passive transepithelial absorption of FITC‐dextran (B), was used as measures of epithelial barrier integrity. Sham adoptive transferred B6 Rag1−/−mice (white bars) were used as baseline controls in some cases. n = 5 mice/group. Data represent three individual experiments and are shown as mean ± SEM. *P 0.05, **P 0.01. Black asterisks compare NCK2187 to PBS‐treated adoptively transferred mice, and red asterisks to NCK56‐treated mice.


A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187 , colonic gene expression of C ‐ type lectin receptors were measured by RT - PCR . Each box represents an mouse ; n = 4 . Data represent three experiments .

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A B6 mice were orally gavaged with 109CFU NCK56 or NCK2187, and the colonic gene expression of C‐type lectin receptors were measured by RT-PCR. Each box represents an individual mouse; n = 4. Data represent three individual experiments.


B Binding of SlpA to various hFc fusion was analyzed by flow cytometry . Gray tinted = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion ; blue = SlpA ‐ coated beads + SIGNR1 ‐ hFc ; red = SlpA ‐ coated beads + SIGNR3 ‐ hFc . Binding results were confirmed five independent times .

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

B Binding of SlpA to various hFc fusion proteins was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; blue = SlpA‐coated beads + SIGNR1‐hFc; red = SlpA‐coated beads + SIGNR3‐hFc. Binding assay results were confirmed five independent times.


C Binding of SlpA to SIGNR3 , but not SIGNR1 expressed by CHO ‐ S . Gray tinted = untransfected CHO ‐ S ; blue = untransfected CHO ‐ S + labeled SlpA ; green = SIGNR1 ‐ transfected CHO ‐ S + labeled SlpA ; red = SIGNR3 ‐ transfected CHO ‐ S + labeled SlpA . Binding assays in CHO ‐ S were performed three times .

O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

C Binding of SlpA to SIGNR3, but not SIGNR1 expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; green = SIGNR1‐transfected CHO‐S cells + labeled SlpA; red = SIGNR3‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


D Binding of SlpA to recombinant human DC ‐ SIGN / CD209 ‐ Fc chimera was analyzed by flow cytometry . Gray tinted = SlpA ‐ coated beads only ; orange = SlpA ‐ coated beads + secondary antibody ; green = SlpA ‐ coated beads + control fusion ; red = SlpA ‐ coated beads + DC ‐ SIGN ‐ Fc . Binding assays were performed confirmed three times .

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

D Binding of SlpA to recombinant human DC‐SIGN/CD209‐Fc chimera was analyzed by flow cytometry. Gray tinted line = SlpA‐coated beads only; orange = SlpA‐coated beads + secondary antibody; green = SlpA‐coated beads + control fusion protein; red = SlpA‐coated beads + DC‐SIGN‐Fc. Binding assays were performed and confirmed three times.


E Binding of SlpA to DC ‐ SIGN expressed by CHO ‐ S . Gray tinted = untransfected CHO ‐ S ; blue = untransfected CHO ‐ S + labeled SlpA ; red = DC ‐ SIGN ‐ transfected CHO ‐ S + labeled SlpA . Binding assays in CHO ‐ S were performed three times .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

E Binding of SlpA to DC‐SIGN expressed by CHO‐S cells. Gray tinted line = untransfected CHO‐S cells; blue = untransfected CHO‐S cells + labeled SlpA; red = DC‐SIGN‐transfected CHO‐S cells + labeled SlpA. Binding assays in CHO‐S cells were performed three individual times.


F , G IL ‐ 1β production by colonic DCs of naïve WT B6 GF or B6 Signr3 − / − ( KO ) mice treated with NCK56 ( blue ) or NCK2187 ( green ) four times , or left untreated ( black ) , as determined by flow cytometry . n = 5 mice / group . Data represent four experiments are shown as mean ± . * P 0 . 05 . Black asterisks compare NCK2187 to untreated ( PBS ) mice , red asterisks to NCK56 ‐ treated mice .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O B-ORGANISM O

F, G IL‐1β production by colonic DCs of naïve WT B6 GF or B6 Signr3−/− (KO) mice treated with NCK56 (blue) or NCK2187 (green) four times, or left untreated (black), as determined by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM. *P 0.05. Black asterisks compare NCK2187 to untreated (PBS) mice, and red asterisks to NCK56‐treated mice.


H Frequency of colonic FoxP3 + Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry . n = 5 mice / group . Data represent four experiments are shown as mean ± .

O O O B-TISSUE B-GENEPROD O B-CELL_TYPE O O B-ORGANISM O O B-ORGANISM O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O

H Frequency of colonic FoxP3+ Tregs in KO mice treated with NCK56 or NCK2187 was measured by flow cytometry. n = 5 mice/group. Data represent four individual experiments and are shown as mean ± SEM.


WT B6 or B6 Signr3 − / − ( KO ) mice were orally gavaged with NCK56 , NCK2187 , or SlpA on days − 3 − 1 , 3 % DSS was given in drinking water . Mice were gavaged with or purified SlpA every other day for an additional three times monitored for disease progression . A Colitis severity was determined in part by loss . n = 5 mice / group .

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

WT B6 or B6 Signr3−/− (KO) mice were orally gavaged with NCK56, NCK2187, or SlpA on days −3 and −1, and 3% DSS was given in the drinking water. Mice were gavaged with bacteria or purified SlpA every other day for an additional three times and monitored for disease progression.A Colitis severity was determined in part by weight loss. n = 5 mice/group.