( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O 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), 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the presence of increasing concentrations of WT or G / S LRRK2 . Inset : representative immunoblot n = 4 - 6 .

O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 protein added .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 the indicated concentrations of trypsin . LAMP - 1 is shown to confirm the integrity of the lysosomes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 the IP ( IP L2A ) corrected for the amount of LRRK2 pulled down and expressed as a multiple of WT value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g,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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 the same samples .

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


( 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 .

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 cells expressing GFP - LRRK2 proteins and pulsed for 24 h with doxycycline . Inputs ( Inp ) , one - quarter of material added .

O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 : quantification of α - syn oligomers , expressed as a multiple of amounts in samples without LRRK2 ( n = 4 or 5 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

O 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 O O O O O O O O O O O O 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-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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) 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 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 .

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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 ) GFP tagged Syb protein expressed in Hh - receiving cells co - localizes with endogenous immuno - labelled Ptc along the apico - basal axis ( 3D reconstruction ) . Vesicles containing Syb and Ptc are visualized at lateral and basal planes ( arrows ) , and can be detected along cytonemes marked with Syb - GFP ( arrowhead ) .

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A) GFP tagged Syb protein expressed in Hh-receiving cells co-localizes with endogenous immuno-labelled Ptc along the apico-basal axis (3D reconstruction). Vesicles containing Syb and Ptc are visualized at lateral and basal planes (arrows), and can be detected along cytonemes marked with Syb-GFP (arrowhead).


B ) Expression of the GFP tagged Syt1 also co - localizes with inmmuno - labelled endogenous Ptc at vesicles ( lateral plane , arrows point at vesicles with co - localization ) and decorates the cytoneme membranes ( basal plane , arrowheads indicate cytonemes ) .

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B) Expression of the GFP tagged Syt1 also co-localizes with inmmuno-labelled endogenous Ptc at vesicles (lateral plane, arrows point at vesicles with co-localization) and decorates the cytoneme membranes (basal plane, arrowheads indicate cytonemes).


C ) GRASP experiment shows exocytic events in Hh - receiving cells at basal membrane contacts . Hh - receiving cells express the GFP barrel 1 - 10 tagged to Syb and Hh - producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4 . Note the fluorescent discrete punctae ( arrows ) along Hh producing cells cytonemes ( stabilized with Ihog - RFP ) crossing the reception area .

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C) GRASP experiment shows exocytic events in Hh-receiving cells at basal membrane contacts. Hh-receiving cells express the GFP barrel 1-10 tagged to Syb and Hh-producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4. Note the fluorescent discrete punctae (arrows) along Hh producing cells cytonemes (stabilized with Ihog-RFP) crossing the reception area.


D ) Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP . Note a strong localization in clusters close to the basal membrane in the 3D reconstruction ( left panel ) and in confocal lateral and basal planes , including cytonemes ( right panels , arrows and arrowhead indicate vesicle - like structures along cytoenemes ) .

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D) Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP. Note a strong localization in clusters close to the basal membrane in the 3D reconstruction (left panel) and in confocal lateral and basal planes, including cytonemes (right panels, arrows and arrowhead indicate vesicle-like structures along cytoenemes).


A ) Ptc protein distribution in a wing disc ( 3D reconstitution ) under down - regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment ( D ) , keeping the ventral compartment ( V ) as a WT internal control . Note the basal accumulation of endogenous Ptc ( arrowheads ) when knocking down Syb or Syt1 .

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A) Ptc protein distribution in a wing disc (3D reconstitution) under down-regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment (D), keeping the ventral compartment (V) as a WT internal control. Note the basal accumulation of endogenous Ptc (arrowheads) when knocking down Syb or Syt1.


B ) Confocal images of immuno - labelled endogenous Ptc and cytonemes stabilized with Ihog - RFP protruding from Hh producing cells in wild type receiving cells ( left panel ) , or B ' ) when either blocking exocytosis by down - regulating Syb ( middle panel ) or B ' ' ) blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin , Shibire ( right panel ) in the Hh - receiving cells . Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization and processing after Hh reception ; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae ( arrowheads ) , and endocytosis inhibition leads to Ptc accumulation at the plasma membrane ( arrows ) .

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B) Confocal images of immuno-labelled endogenous Ptc and cytonemes stabilized with Ihog-RFP protruding from Hh producing cells in wild type receiving cells (left panel), or B') when either blocking exocytosis by down-regulating Syb (middle panel) or B'') blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin, Shibire (right panel) in the Hh-receiving cells. Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization and processing after Hh reception; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae (arrowheads), and endocytosis inhibition leads to Ptc accumulation at the plasma membrane (arrows).


C ) Confocal apical ( left ) and basal ( right ) images of a shits mutant wing disc expressing Syb RNAi dorsally ( D ) to also block exocytosis , keeping the ventral compartment ( V ) as an internal control where just endocytosis is inhibited . After dorsal Syb RNAi induction , endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature . Note the dorsal reduction of Ptc at plasma membrane after blocking exocytosis .

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C) Confocal apical (left) and basal (right) images of a shits mutant wing disc expressing Syb RNAi dorsally (D) to also block exocytosis, keeping the ventral compartment (V) as an internal control where just endocytosis is inhibited. After dorsal Syb RNAi induction, endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature. Note the dorsal reduction of Ptc at plasma membrane after blocking exocytosis.


D ) Quantification of Ptc - GFP levels ( Yac construct ) along the wing disc apico - basal axis , integrating both fluorescence intensity and signal area ( Integrated Density ) . The graph on the left shows values for wild type situations in purple ( average in blue ) , while Syb RNAi treatment values are shown in green ( average in orange ) . a clear shift of higher values towards the basal side of the discs after inhibition of Syb function . The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side ( - 1 = basal , + 1 = apical ) of discs after RNAi expression for different SNARE proteins and the wild type . Central horizontal lines show median values of N = 7 - 16 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . A basal association is particularly noticeable for Syb , Syx1A and α - Snap down - regulation . Right panel is a scheme illustrating the wild type and RNAi - treated patterns of quantified Ptc - GFP levels distribution along the wing disc apico - basal axis .

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D) Quantification of Ptc-GFP levels (Yac construct) along the wing disc apico-basal axis, integrating both fluorescence intensity and signal area (Integrated Density). The graph on the left shows values for wild type situations in purple (average in blue), while Syb RNAi treatment values are shown in green (average in orange). a clear shift of higher values towards the basal side of the discs after inhibition of Syb function. The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side (-1=basal, +1=apical) of discs after RNAi expression for different SNARE proteins and the wild type. Central horizontal lines show median values of N=7-16, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. A basal association is particularly noticeable for Syb, Syx1A and α-Snap down-regulation. Right panel is a scheme illustrating the wild type and RNAi-treated patterns of quantified Ptc-GFP levels distribution along the wing disc apico-basal axis.


A ) Electron Microscopy imaging of wing discs expressing the UAS - Ptc - GFP construct for 24 hours in receiving cells using Ptc - Gal4 ; TubGal80ts . Left panel shows areas with anti - GFP immuno - labelling ( marked in blue ) throughout the apico / basal axis of the wing disc epithelium a ) anti - GFP gold - labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation . b ) Ptc immuno - gold labelling is present in multivesicular bodies ( MVBs ) subapically ( top right panels ) . b1 ) subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes . c ) Ptc label is also present in MVBs close to the basal membrane ( lower right panels ) , c1 ) basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles ( ILV ) .

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A) Electron Microscopy imaging of wing discs expressing the UAS-Ptc-GFP construct for 24 hours in receiving cells using Ptc-Gal4; TubGal80ts. Left panel shows areas with anti-GFP immuno-labelling (marked in blue) throughout the apico/basal axis of the wing disc epithelium a) anti-GFP gold-labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation. b) Ptc immuno-gold labelling is present in multivesicular bodies (MVBs) subapically (top right panels). b1) subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes. c) Ptc label is also present in MVBs close to the basal membrane (lower right panels), c1) basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles (ILV).


B ) Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5 . The top panel is a schematic representation of the localized expression . The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections . that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc , compared to the wild type control on the ventral side .

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B) Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5. The top panel is a schematic representation of the localized expression. The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections. that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side.


accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc , compared to the wild type control on the ventral side . C ) Digital 3D reconstruction ( left ) and apical and basal confocal sections ( right ) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire .

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accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side. C) Digital 3D reconstruction (left) and apical and basal confocal sections (right) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire.


A ) 3D reconstructions of immuno - labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components . All RNAis ' expression result in Ptc accumulation in large structures , located apically under Hrs ( ESCRT - 0 component ) inhibition , and basally under inhibition of the ESCRT components I ( Tsg101 ) , II ( Vps22 ) and III ( Shrub ) . Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation .

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A) 3D reconstructions of immuno-labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components. All RNAis' expression result in Ptc accumulation in large structures, located apically under Hrs (ESCRT-0 component) inhibition, and basally under inhibition of the ESCRT components I (Tsg101), II (Vps22) and III (Shrub). Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation.


B ) Detailed confocal imaging of immuno - labelled endogenous Ptc localization after clonal Tsg101 down - regulation . The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells ( arrows ) , since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh

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B) Detailed confocal imaging of immuno-labelled endogenous Ptc localization after clonal Tsg101 down-regulation. The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells (arrows), since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh reception


C ) Ptc accumulation in these punctated structures ( arrows ) is also visualized on receiving cytonemes stabilized with the co - receptor Ihog - RFP and after Tsg101 inhibition .

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C) Ptc accumulation in these punctated structures (arrows) is also visualized on receiving cytonemes stabilized with the co-receptor Ihog-RFP and after Tsg101 inhibition.


D ) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog - RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions ( arrow ) .

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D) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog-RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions (arrow).


A ) Confocal image of wing disc expressing the human tetraspanin CD63 - GFP expressed in the Hh receiving cells colocalizing with Ptc ( Bac . Ptc - cherry ) in vesicle - like structures ( yellow arrows ) along basal cytonemes .

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A) Confocal image of wing disc expressing the human tetraspanin CD63-GFP expressed in the Hh receiving cells colocalizing with Ptc (Bac.Ptc-cherry) in vesicle-like structures (yellow arrows) along basal cytonemes.


B ) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63 - Cherry and Ptc ( Yac . Ptc - GFP ) at punctae moving in anterograde direction along cytonemes ( white arrowheads show trajectory in time - lapse frames ) .

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B) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63-Cherry and Ptc (Yac.Ptc-GFP) at punctae moving in anterograde direction along cytonemes (white arrowheads show trajectory in time-lapse frames).


C ) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F - RFP ( orthologous to the mammalian tetraspanin CD9 / CD81 ) ( yellow arrows indicate co - localization in punctate structures ) .

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C) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F-RFP (orthologous to the mammalian tetraspanin CD9/CD81) (yellow arrows indicate co-localization in punctate structures).


D ) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F - RFP in the Hh receiving cells shows colocalization with Ptc - GFP at mobile punctae ( yellow arrows ) .

D) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F-RFP in the Hh receiving cells shows colocalization with Ptc-GFP at mobile punctae (yellow arrows).


E ) Tsp96F - RFP expressed in wing disc Hh - receiving cells also shows localization in basal cytonemes stabilized by Ihog - CFP expression ( yellow arrows indicate Tsp96F - RFP - positive punctate structures ) .

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E) Tsp96F-RFP expressed in wing disc Hh-receiving cells also shows localization in basal cytonemes stabilized by Ihog-CFP expression (yellow arrows indicate Tsp96F-RFP-positive punctate structures).


F ) Frames showing in vivo localization of Tsp96 - RFP punctae to cytonemes stabilized with Ihog - CFP expressed in the abdominal histoblasts Hh - receiving cells , moving in an anterograde fashion towards the P compartment ( white arrowheads show trajectory in time - lapse frames ) .

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F) Frames showing in vivo localization of Tsp96-RFP punctae to cytonemes stabilized with Ihog-CFP expressed in the abdominal histoblasts Hh-receiving cells, moving in an anterograde fashion towards the P compartment (white arrowheads show trajectory in time-lapse frames).


G ) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co - localization with endogenous Ptc at cytonemes ( yellow arrows indicate Tsp96F - HA and Ptc co - localizing punctate structures ) .

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G) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co-localization with endogenous Ptc at cytonemes (yellow arrows indicate Tsp96F-HA and Ptc co-localizing punctate structures).


H ) Western blot against HA - tag of Immuno - Precipitated sample ( IP ) , Depleted Sample ( Dep ) , High Speed Sample ( HSS ) Whole Sample and Control sample from a co - immunoprecipitation experiment isolating GFP of Tsp96F - HA and Ptc - GFP expressing larvae or GFP and Tsp96F - HA expressing larvae as negative control . Arrowhead indicates the positive band corresponding to Tsp96F - HA expected molecular weight ( 30 - 40 kDa ) , present in all samples including the immuno - precipitated one and not present in the negative control sample .

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H) Western blot against HA-tag of Immuno-Precipitated sample (IP), Depleted Sample (Dep), High Speed Sample (HSS) Whole Sample and Control sample from a co-immunoprecipitation experiment isolating GFP of Tsp96F-HA and Ptc-GFP expressing larvae or GFP and Tsp96F-HA expressing larvae as negative control. Arrowhead indicates the positive band corresponding to Tsp96F-HA expected molecular weight (30-40 kDa), present in all samples including the immuno-precipitated one and not present in the negative control sample.


A ) 3D reconstruction of wing disc immuno - labelled for endogenous Ptc and dorsally over - expressing the Tsp96F - HA construct , keeping the ventral side of the disc in wild type condition as internal control . the dorsal accumulation of Ptc in very large punctae across the apico - basal axis , but mainly at the basal side of the epithelium . On the right , graphs represent fluorescence values from apical to basal , comparing dorsal and ventral sides and showing a shift of the highest values towards the basal side after Tsp96 - HA expression .

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A) 3D reconstruction of wing disc immuno-labelled for endogenous Ptc and dorsally over-expressing the Tsp96F-HA construct, keeping the ventral side of the disc in wild type condition as internal control. the dorsal accumulation of Ptc in very large punctae across the apico-basal axis, but mainly at the basal side of the epithelium. On the right, graphs represent fluorescence values from apical to basal, comparing dorsal and ventral sides and showing a shift of the highest values towards the basal side after Tsp96-HA expression.


B ) Confocal image of a wing disc with a clone expressing Tsp96F - HA and immuno - labelled with anti - Ptc and anti - HA antibodies , which co - localize in the large basolateral punctae ( yellow arrows ) .

B) Confocal image of a wing disc with a clone expressing Tsp96F-HA and immuno-labelled with anti-Ptc and anti-HA antibodies, which co-localize in the large basolateral punctae (yellow arrows).


C ) Electron microscopy image from immunolabelled ( anti - GFP ) Hh - receiving cells of a wing disc expressing Ptc - GFP and Tsp96F - HA . The detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc .

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C) Electron microscopy image from immunolabelled (anti-GFP) Hh-receiving cells of a wing disc expressing Ptc-GFP and Tsp96F-HA. The detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc.


A ) Ptc is mainly secreted in small EVs ( P100K ) from Cl8 cells .

A) Ptc is mainly secreted in small EVs (P100K) from Cl8 cells.


B ) Large EVs ( lEVs ) , small EVs ( sEVs ) and very small EVs ( vsEVs ) were analysed in triplicate by Western blotting . sEVs are enriched in Ptc and in the EV - associated proteins Syntaxin1 ( Synt1A ) , Late bloomer ( Lbm ) and Hsp70 .

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B) Large EVs (lEVs), small EVs (sEVs) and very small EVs (vsEVs) were analysed in triplicate by Western blotting. sEVs are enriched in Ptc and in the EV-associated proteins Syntaxin1 (Synt1A), Late bloomer (Lbm) and Hsp70.


C ) Fractionation of sEVs by Sucrose Density Gradient : Ptc partially co - fractioned with Synt1A , Lbm and Hsp70 . Note that while unprocessed form of co - receptor Ihog does not co - fraction with Ptc , the processed form does in the same subpopulation .

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C) Fractionation of sEVs by Sucrose Density Gradient: Ptc partially co-fractioned with Synt1A, Lbm and Hsp70. Note that while unprocessed form of co-receptor Ihog does not co-fraction with Ptc, the processed form does in the same subpopulation.


D ) Fractionation of sEVs by Size Exclusion Chromatography : Ptc perfectly co - fractioned with Synt1A , Lbm and Hsp70 . In agreement with the density gradient analysis , unprocessed form of co - receptor Ihog does not co - fraction with Ptc in the same subpopulation of sEVs .

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D) Fractionation of sEVs by Size Exclusion Chromatography: Ptc perfectly co-fractioned with Synt1A, Lbm and Hsp70. In agreement with the density gradient analysis, unprocessed form of co-receptor Ihog does not co-fraction with Ptc in the same subpopulation of sEVs.


E ) Effect on EV - associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α - Snap , the tetraspanin Tsp96F and the ESCRT components Hrs , Tsg101 , Shrub and Vps4 . Hsp70 in cells and EVs is detected by different exposition time .

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E) Effect on EV-associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α-Snap, the tetraspanin Tsp96F and the ESCRT components Hrs, Tsg101, Shrub and Vps4. Hsp70 in cells and EVs is detected by different exposition time.


A ) Projection images of three confocal sections ( 1 μm ) at the basal side of wing discs expressing the Bac constructs for Hh - GFP and Ptc - cherry , used to analyse co - localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion ( Syb RNAi ) , vesicle trafficking ( Hrs RNAi ) or Ptc protein degradation ( Dor RNAi ) . Boxplot shows co - localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4 , SNAREs Syb and α - Snap , and Dor as control for effects after Ptc degradation - blocking , as well as temporally over - expressing the Tsp96F - HA . Central horizontal lines show median values of N = 8 - 12 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding . Note the significant reduction in co - localization rates ( 1 = total coverage of red by green ) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F - HA over - expression , while RNAi expression for Dor presents greater co - localization .

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A) Projection images of three confocal sections (1 μm) at the basal side of wing discs expressing the Bac constructs for Hh-GFP and Ptc-cherry, used to analyse co-localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion (Syb RNAi), vesicle trafficking (Hrs RNAi) or Ptc protein degradation (Dor RNAi). Boxplot shows co-localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4, SNAREs Syb and α-Snap, and Dor as control for effects after Ptc degradation-blocking, as well as temporally over-expressing the Tsp96F-HA. Central horizontal lines show median values of N=8-12 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding. Note the significant reduction in co-localization rates (1= total coverage of red by green) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F-HA over-expression, while RNAi expression for Dor presents greater co-localization.


B ) Projection images of three confocal sections ( 1 μm ) from the apical side of the wing discs . Quantification of co - localization rates using apical projection images ; central horizontal lines show median values of N = 8 - 12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note that at the apical side no clear significant reduction in co - localization rates ( 1 = total coverage of red by green ) is observed after RNAi expression or in Tsp96F - HA over - expression .

B) Projection images of three confocal sections (1 μm) from the apical side of the wing discs. Quantification of co-localization rates using apical projection images; central horizontal lines show median values of N=8-12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note that at the apical side no clear significant reduction in co-localization rates (1= total coverage of red by green) is observed after RNAi expression or in Tsp96F-HA over-expression.


C ) Correlation coefficients between the maximum value of quantified integrated density ( integrating both fluorescence intensity and signal area ) for Ptc - GFP values ( Yac construct ) and distance from the basal side ( - 1 = basal , + 1 = apical ) . Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor ( necessary for Ptc degradation ) , the ESCRT Hrs , the SNARE Syb as well as after over - expression of the Tsp96F - HA construct and wild type condition . Central horizontal lines show median values of N = 8 - 12 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note the apical localization of Ptc - GFP highest values after degradation impairment ( Dor RNAi ) in contrast to the basal shift resulting from exocytosis deficiency ( Syb RNAi ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation coefficients between the maximum value of quantified integrated density (integrating both fluorescence intensity and signal area) for Ptc-GFP values (Yac construct) and distance from the basal side (-1=basal, +1=apical). Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor (necessary for Ptc degradation), the ESCRT Hrs, the SNARE Syb as well as after over-expression of the Tsp96F-HA construct and wild type condition. Central horizontal lines show median values of N=8-12, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note the apical localization of Ptc-GFP highest values after degradation impairment (Dor RNAi) in contrast to the basal shift resulting from exocytosis deficiency (Syb RNAi).


A ) Confocal images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier ( Col ) of wild type wing discs and discs after inhibition of vesicle fusion ( α - Snap RNAi ) , vesicle trafficking ( Hrs RNAi ) or in over - expression of the Tsp96F - HA . Quantification of gradient lengths ( μm ) using Collier signal and normalized to the wing pouch size , showing the proportion of wing pouch covered by signal ( 1 = total coverage ) . Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs , Tsg101 , Shrub and Vps4 and for the SNARE proteins Syb , Snap24 , α - Snap , as well as in transient over - expression of the Tsp96F - HA . Central horizontal lines show median values of N = 7 - 22 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier (Col) of wild type wing discs and discs after inhibition of vesicle fusion (α-Snap RNAi), vesicle trafficking (Hrs RNAi) or in over-expression of the Tsp96F-HA. Quantification of gradient lengths (μm) using Collier signal and normalized to the wing pouch size, showing the proportion of wing pouch covered by signal (1=total coverage). Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs, Tsg101, Shrub and Vps4 and for the SNARE proteins Syb, Snap24, α-Snap, as well as in transient over-expression of the Tsp96F-HA. Central horizontal lines show median values of N=7-22 , box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition.


B ) Lateral confocal images and quantification of the extension of immuno - labeled Engrailed as the high threshold Hh target . Quantification shows number of cell diameters covered by signal from the posterior / anterior border marked by Hh - GFP ( Bac construct ) ; central horizontal lines show median values of N = 6 - 10 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers . Note a significant reduction after RNAi expression , suggesting Hh reception impairment and flattening of the signal gradient .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lateral confocal images and quantification of the extension of immuno-labeled Engrailed as the high threshold Hh target. Quantification shows number of cell diameters covered by signal from the posterior/anterior border marked by Hh-GFP (Bac construct); central horizontal lines show median values of N=6-10 box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a significant reduction after RNAi expression, suggesting Hh reception impairment and flattening of the signal gradient.


C ) Lateral confocal images and quantification of Ptc transcription as a Hh target through immunolabeling of the reporter β − galactosidase ( Ptc - LacZ ) . Middle graph shows the extension covered by signal normalized to wing pouch size , in wild type condition ( N = 6 ) or after ectopic expression of the Tsp96F - HA construct ( N = 9 ) . Green shaded areas show data variability and dotted line gives the average values . Note the greater distance covered after Tsp96F - HA transient expression . Boxplot ( right ) shows distance quantifications with central horizontal lines showing median values of N = 6 - 9 , box showing lower and upper quartiles and whiskers showing the maximum and minimum excluding outliers . Note a significant extension under Tsp96F - HA expression , suggesting again flattening of the signal gradient .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lateral confocal images and quantification of Ptc transcription as a Hh target through immunolabeling of the reporter β−galactosidase (Ptc-LacZ). Middle graph shows the extension covered by signal normalized to wing pouch size, in wild type condition (N=6) or after ectopic expression of the Tsp96F-HA construct (N=9). Green shaded areas show data variability and dotted line gives the average values. Note the greater distance covered after Tsp96F-HA transient expression. Boxplot (right) shows distance quantifications with central horizontal lines showing median values of N=6-9 , box showing lower and upper quartiles and whiskers showing the maximum and minimum excluding outliers. Note a significant extension under Tsp96F-HA expression, suggesting again flattening of the signal gradient.


C . Representative flow cytometric profiles of thymocytes from 6 - week old Rb ∆ K4 and control mice for CD3 - , CD4 - and CD8 - positive cell populations . Bottom , average of CD3 - , CD4 - and CD8 single - and double - positive cells in Rb ∆ K4 vs . control thymocytes showing no significant ( n . s . ) difference by student ' s t - test . Bars represent mean ± SD ( standard deviation ) ; n = 6 biological replicates ) .

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C. Representative flow cytometric profiles of thymocytes from 6-week old Rb∆K4 and control mice for CD3-, CD4- and CD8-positive cell populations. Bottom, average of CD3-, CD4- and CD8 single- and double-positive cells in Rb∆K4 vs. control thymocytes showing no significant (n.s.) difference by student's t-test. Bars represent mean ±SD (standard deviation); n=6 biological replicates).


D . Short telomeres in Rb ∆ K4 mice . Images of splenocyte metaphase spreads hybridized with the telomere repeat probe ( pink ) . Note shorter telomeres in Rb ∆ K4 splenocytes ( arrows ) . Bottom , quantitative analysis of 10 splenocyte metaphases from one pair of Rb ∆ K4 and control littermate ; ( P < 0 . 001 by two - tailed Mann - Whitney - Wilcoxon test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Short telomeres in Rb∆K4 mice. Images of splenocyte metaphase spreads hybridized with the telomere repeat probe (pink). Note shorter telomeres in Rb∆K4 splenocytes (arrows). Bottom, quantitative analysis of 10 splenocyte metaphases from one pair of Rb∆K4 and control littermate; (P<0.001 by two-tailed Mann-Whitney-Wilcoxon test).


C . Left , representative flow cytometric analysis for CD3 , CD4 and CD8 , showing near normal T cell development in 10 - day - old Rb ∆ K7 mice . Right , average of CD3 , CD4 and CD8 single - and double - positive cell populations in Rb ∆ K7 vs control thymocytes with no significant difference ( n . s . ) by student ' s t - test . Results are presented as mean ± SD , n = 4 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left, representative flow cytometric analysis for CD3, CD4 and CD8, showing near normal T cell development in 10-day-old Rb∆K7 mice. Right, average of CD3, CD4 and CD8 single- and double-positive cell populations in Rb∆K7 vs control thymocytes with no significant difference (n.s.) by student's t-test. Results are presented as mean ±SD, n=4 biological replicates).


D . Left , images showing apparent normal division of retinal progenitors in Rb ∆ K7 mice . Mice were labeled with BrdU for 2 hrs ; retinal sections from indicated genotypes were stained with DAPI ( blue ) , Ki67 ( green ) , PH3 ( red ) or BrdU ( red ) to mark nuclei , all dividing cells , mitotic or S - phase cells , respectively . Right , quantification of staining showing no significant difference among the genotypes . Bars denote mean ± SD ; n = 3 biological replicates ) . Scale bar : 20 μm .

D. Left, images showing apparent normal division of retinal progenitors in Rb∆K7 mice. Mice were labeled with BrdU for 2 hrs; retinal sections from indicated genotypes were stained with DAPI (blue), Ki67 (green), PH3 (red) or BrdU (red) to mark nuclei, all dividing cells, mitotic or S-phase cells, respectively. Right, quantification of staining showing no significant difference among the genotypes. Bars denote mean ±SD; n=3 biological replicates). Scale bar: 20 μm.


E . Left , top , western blot analysis of Rb ∆ K7 thymocytes with an antibody that recognizes both hyper - phosphorylated ( ppRb ) and hypo - phosphorylated pRb species . Right , phospho - specific immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in Rb ∆ K7 thymocytes . Right , immuno - precipitation ( IP ) - Western blot analysis demonstrating phosphorylation of pRb ∆ K7 on Ser243 / Thr246 ( top ) and Ser605 ( bottom ) . n . s . , non - specific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Left, top, western blot analysis of Rb∆K7 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. Right, phospho-specific immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in Rb∆K7 thymocytes. Right, immuno-precipitation (IP)-Western blot analysis demonstrating phosphorylation of pRb∆K7 on Ser243/Thr246 (top) and Ser605 (bottom). n.s., non-specific band.


B . Increased resistance of Rb ∆ K7 vs . control MEFs to CDK2 inhibition ( P = 0 . 008 by two - tailed unpaired student ' s t - test ) . Effect of a pan CDK inhibitor was near significance ( P = 0 . 07 ) . Bars represent mean ± SD , n = 3 independent MEFs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Increased resistance of Rb∆K7 vs. control MEFs to CDK2 inhibition (P=0.008 by two-tailed unpaired student's t-test). Effect of a pan CDK inhibitor was near significance (P=0.07). Bars represent mean ±SD, n=3 independent MEFs.


C . Left , representative flow cytometric analysis with 7 - AAD and AnnexinV , showing a significant decrease in apoptosis in Rb ∆ K7 MEFs relative to control . Right , significant differences in average percentage of live ( 7 - AAD - : AnnexinV - ) vs . apoptotic ( 7 - AAD + : AnnexinV + ) cells . Bars represent mean ± SD ; P value calculated by two - tailed unpaired student ' s t - test ; n = 3 independent MEFs .

C. Left, representative flow cytometric analysis with 7-AAD and AnnexinV, showing a significant decrease in apoptosis in Rb∆K7 MEFs relative to control. Right, significant differences in average percentage of live (7-AAD-:AnnexinV-) vs. apoptotic (7-AAD+:AnnexinV+) cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test; n=3 independent MEFs.


F - I . Delayed hair re - growth in eight - month old Rb ∆ K7 mice two weeks post hair plucking ( F ) . ( G ) Abnormal skin histology in Rb ∆ K7 mice relative to control . FCL , subcutaneous fat cell layer , T , telogen ; A , anagen . ( H - I ) Representative immunostaining for keratin 14 and PCNA showing reduced expression in Rb ∆ K7 hair follicles vs controls . Scale bar , 50 μm .

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F-I. Delayed hair re-growth in eight-month old Rb∆K7 mice two weeks post hair plucking (F). (G) Abnormal skin histology in Rb∆K7 mice relative to control. FCL, subcutaneous fat cell layer, T, telogen; A, anagen. (H-I) Representative immunostaining for keratin 14 and PCNA showing reduced expression in Rb∆K7 hair follicles vs controls. Scale bar, 50 μm.


J . Hyperglycemia in Rb ∆ K7 but not Rb ∆ K4 mice . Blood glucose in 10 - 12 - month old fasting Rb ∆ K7 ( left , n = 11 ) or 18 - month old fasting Rb ∆ K4 ( right , n = 15 ) mice versus control littermates ( n = 6 and n = 8 , respectively ) . Mean ± SD ; P values by two - tailed unpaired student ' s t - test .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Hyperglycemia in Rb∆K7 but not Rb∆K4 mice. Blood glucose in 10-12-month old fasting Rb∆K7 (left, n=11) or 18-month old fasting Rb∆K4 (right, n=15) mice versus control littermates (n=6 and n=8, respectively). Mean ±SD; P values by two-tailed unpaired student's t-test.


B . Glucose tolerance Test ( GTT ) demonstrating progressive defect in clearance of blood glucose in Rb ∆ K7 mice . 6 - 7 week ( left ) or 6 month ( right ) old fasting Rb ∆ K7 mice and control littermates ( n = 5 - 6 each per group ) were injected with glucose , and blood glucose was determined at indicated intervals . Error bars represent SD .

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 O O O O O O O O O O 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

B. Glucose tolerance Test (GTT) demonstrating progressive defect in clearance of blood glucose in Rb∆K7 mice. 6-7 week (left) or 6 month (right) old fasting Rb∆K7 mice and control littermates (n=5-6 each per group) were injected with glucose, and blood glucose was determined at indicated intervals. Error bars represent SD.


F . Loss of pancreatic islet area in Rb ∆ K7 mice correlates with reduced proliferation and H3K36me3 histone marks , but not with increased cell death . TUNEL analysis showing apoptosis in 10 day - old Rb ∆ K7 islets is even lower than in control littermates . Arrow points to a rare apoptotic cell in control islet . Serine800 / 804 phosphorylation of pRb in β - cells from 10 day - old control but not Rb ∆ K7 islets with a Ser - to - Ala substitution at this site . BrdU incorporation and PH3 staining showing reduced S - phase and mitotic cells , respectively , in 2 - and 3 - week old Rb ∆ K7 vs control islets . Reduced H3K36me3 staining in pancreatic islets from 3 - week old Rb ∆ K7 mice compared to control littermates ( n > 3 for each experiment ) . Scale bar , 100 μm .

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F. Loss of pancreatic islet area in Rb∆K7 mice correlates with reduced proliferation and H3K36me3 histone marks, but not with increased cell death. TUNEL analysis showing apoptosis in 10 day-old Rb∆K7 islets is even lower than in control littermates. Arrow points to a rare apoptotic cell in control islet. Serine800/804 phosphorylation of pRb in β-cells from 10 day-old control but not Rb∆K7 islets with a Ser-to-Ala substitution at this site. BrdU incorporation and PH3 staining showing reduced S-phase and mitotic cells, respectively, in 2- and 3-week old Rb∆K7 vs control islets. Reduced H3K36me3 staining in pancreatic islets from 3-week old Rb∆K7 mice compared to control littermates (n>3 for each experiment). Scale bar, 100 μm.


H . Reduced ability of Rb ∆ K7 β - cell to re - enter the cell cycle in response to pregnancy ( top ) or mitogenic ( bottom ) signals . Top , cell proliferation in P14 . 5 pregnant Rb ∆ K7 islet β - cell vs . control was determined by double immunostaining with BrdU and insulin ( left ) ; quantification ( right ) . Results were normalized for the number of BrdU / Insulin positive cells in control islets . An independent experiment using ki67 to quantify cell cycle re - entry top . Bottom , cell proliferation in Rb ∆ K7 β - cell vs . control islets following Exendin4 treatment was determined by immunostaining for Ki67 ; results were normalized for the number of positive cells in control islets . Mean ± SD , P values by two - tailed unpaired student ' s t - test . Scale bar , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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. Reduced ability of Rb∆K7 β-cell to re-enter the cell cycle in response to pregnancy (top) or mitogenic (bottom) signals. Top, cell proliferation in P14.5 pregnant Rb∆K7 islet β-cell vs. control was determined by double immunostaining with BrdU and insulin (left); quantification (right). Results were normalized for the number of BrdU/Insulin positive cells in control islets. An independent experiment using ki67 to quantify cell cycle re-entry top. Bottom, cell proliferation in Rb∆K7 β-cell vs. control islets following Exendin4 treatment was determined by immunostaining for Ki67; results were normalized for the number of positive cells in control islets. Mean ±SD, P values by two-tailed unpaired student's t-test. Scale bar, 100 μm.


I . Representative immunostaining showing induction of PDX1 ( islet ) , SOX9 ( pancreatic epithelium ) and MCM4 ( islet ) expression in Rb ∆ K7 vs control mice . Scale bar , 100 μm .

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I. Representative immunostaining showing induction of PDX1 (islet), SOX9 (pancreatic epithelium) and MCM4 (islet) expression in Rb∆K7 vs control mice. Scale bar, 100 μm.


D . Representative image of a pancreatic islet from a 9 - month old Rb ∆ K7 mouse immunestained for the SASP marker IL - 6 ( red ) and insulin ( green ) . DAPI was used to stain nuclei ( blue ) . Arrows point to IL - 6 + , insulin + pancreatic β - cells . Scale bar , 20 μm .

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D. Representative image of a pancreatic islet from a 9-month old Rb∆K7 mouse immunestained for the SASP marker IL-6 (red) and insulin (green). DAPI was used to stain nuclei (blue). Arrows point to IL-6+, insulin+ pancreatic β-cells. Scale bar, 20 μm.


E . Representative immunostaining for the DDR marker γ - H2A in islets from 3 - week old Rb ∆ K7 and control mice ( left ) ; statistical analysis ( right ; n = 5 each ) . Arrows point to pancreatic γ - H2A - positive Rb ∆ K7 islet cells . Error bars represent SD ; P value calculated by one - way ANOVA , Tukey ' s multiple comparisons test . Scale bar , 100 μm .

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E. Representative immunostaining for the DDR marker γ-H2A in islets from 3-week old Rb∆K7 and control mice (left); statistical analysis (right; n=5 each). Arrows point to pancreatic γ-H2A-positive Rb∆K7 islet cells. Error bars represent SD; P value calculated by one-way ANOVA, Tukey's multiple comparisons test. Scale bar, 100 μm.


H . Vitamin C diet increases 5hmC expression and improves β - cell morphology and islet size in Rb ∆ K7 mice . Scale bar , 100 μm . I . Vitamin C diet reduces the difference in γ − H2A + β - cells in Rb ∆ K7 vs control pancreatic islets relative to regular diet by 3 . 74 folds . Bars indicate mean ± SD . Adjusted P values by one - way ANOVA , Tukey ' s multiple comparisons test . J . Vitamin C diet increases pancreatic islet cell cycle re - entry during pregnancy of Rb ∆ K7 mice relative to control pregnant mice ( no statistical difference , n . s . ) or relative to pregnant Rb ∆ K7 vs control mice fed on regular diet ( P < 0 . 0001 ) by 3 . 8 folds . Bars represent mean ± SD . Adjusted P values by one - way ANOVA , Tukey ' s multiple comparisons test .

O O B-CONTROLLED_VAR I-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 O B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-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 O 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. Vitamin C diet increases 5hmC expression and improves β-cell morphology and islet size in Rb∆K7 mice. Scale bar, 100 μm. I. Vitamin C diet reduces the difference in γ−H2A+ β-cells in Rb∆K7 vs control pancreatic islets relative to regular diet by 3.74 folds. Bars indicate mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test. J. Vitamin C diet increases pancreatic islet cell cycle re-entry during pregnancy of Rb∆K7 mice relative to control pregnant mice (no statistical difference, n.s.) or relative to pregnant Rb∆K7 vs control mice fed on regular diet (P<0.0001) by 3.8 folds. Bars represent mean ±SD. Adjusted P values by one-way ANOVA, Tukey's multiple comparisons test.


B . Left , top , effect of retrovirus - mediated transduction of under - phosphorylated Rb ∆ K11 ( rRb ∆ K11 ) on Rb - deficient mammary tumor cell proliferation as determined by MTT assay ( n = 4 - 5 technical replicates ) . Left , bottom , quantification of SA - βGAL in rRb ∆ K11 - transduced cells . Bars represent mean ± SD ; P value calculated by two - tailed unpaired student ' s t - test . Right , representative images of SA - βGAL staining showing robust senescence ( arrows ) in rRb ∆ K11 - transduced cells . Original magnification : 200x .

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B. Left, top, effect of retrovirus-mediated transduction of under-phosphorylated Rb∆K11 (rRb∆K11) on Rb-deficient mammary tumor cell proliferation as determined by MTT assay (n=4-5 technical replicates). Left, bottom, quantification of SA-βGAL in rRb∆K11-transduced cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test. Right, representative images of SA-βGAL staining showing robust senescence (arrows) in rRb∆K11-transduced cells. Original magnification: 200x.


F . Vitamin C reduces cellular senescence induced by retroviral - mediated over - expression of Rb ∆ K11 . Top - images of SA - βGAL staining in human diploid cells , IMR90 , transduced with control or rRb ∆ K11 virus with or without vitamin C added at the time of infection . Arrows point to SA - βGAL - positive cells . Bottom left , quantification of SA - βGAL staining ( bars represent mean ± SEM ; n = 4 biological replicates ) . P values by one - way ANOVA , Tukey ' s multiple comparisons test . Bottom right , dot - blot analysis with antibody for 5hmC on DNA extracted from IMR90 cells treated or not with vitamin C . Original magnification : 200x .

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F. Vitamin C reduces cellular senescence induced by retroviral-mediated over-expression of Rb∆K11. Top - images of SA-βGAL staining in human diploid cells, IMR90, transduced with control or rRb∆K11 virus with or without vitamin C added at the time of infection. Arrows point to SA-βGAL-positive cells. Bottom left, quantification of SA-βGAL staining (bars represent mean ±SEM; n=4 biological replicates). P values by one-way ANOVA, Tukey's multiple comparisons test. Bottom right, dot-blot analysis with antibody for 5hmC on DNA extracted from IMR90 cells treated or not with vitamin C. Original magnification: 200x.


( K TUNEL staining of testis sections from WT and KO mice at four months of age ( K ) White dotted line indicates the outline of testis tubules .

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(K TUNEL staining of testis sections from WT and KO mice at four months of age (K) White dotted line indicates the outline of testis tubules.


L ) TUNEL staining of testis sections from WT and KO mice at four months of age with apoptotic cell number per tubule ( L ) . n = 3 in ( L ) .

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L) TUNEL staining of testis sections from WT and KO mice at four months of age with apoptotic cell number per tubule (L). n=3 in (L).


( A ) Immunostaining of SYCP3 and γH2AX in sections from adult WT mice . Arrow and arrowhead indicate diffuse and dot - like γH2AX staining , respectively . White dotted line indicates the outline of testis tubules . ( B ) Proportion of tubules of γH2AX expression patterns . At least 100 tubules of each mouse were counted . n = 4 for WT and n = 5 for KO . ( C ) Average number of spermatocytes with γH2AX expression patterns per tubule . At least 50 tubules of each mouse were counted . n = 3 .

(A) Immunostaining of SYCP3 and γH2AX in sections from adult WT mice. Arrow and arrowhead indicate diffuse and dot-like γH2AX staining, respectively. White dotted line indicates the outline of testis tubules. (B) Proportion of tubules of γH2AX expression patterns. At least 100 tubules of each mouse were counted. n=4 for WT and n=5 for KO. (C) Average number of spermatocytes with γH2AX expression patterns per tubule. At least 50 tubules of each mouse were counted. n=3.


( D - H ) Representative nuclear spreads of WT and KO spermatocytes at leptonema ( D ) , zygonema ( E ) , early pachynema ( F ) , late pachynema ( G ) and diplonema ( H ) . Spermatocytes were immunostained for SYCP3 , γH2AX , and H1t . Magnified views are indicated by dashed areas . Schematic representation shows homolog characteristic revealed by SYCP3 signals at different backgrounds and stages . Arrowheads indicate bivalent stretches lacking SYCP3 signals in KO . ( I ) Proportion statistics of spermatocytes with discontinuous SYCP3 signals . At least 200 cells of each mouse were counted . n = 3 for WT and n = 4 for KO . ( J ) Meiotic stage frequencies in ( D - H ) . At least 200 cells of each mouse were counted . n = 3 .

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(D-H) Representative nuclear spreads of WT and KO spermatocytes at leptonema (D), zygonema (E), early pachynema (F), late pachynema (G) and diplonema (H). Spermatocytes were immunostained for SYCP3, γH2AX, and H1t. Magnified views are indicated by dashed areas. Schematic representation shows homolog characteristic revealed by SYCP3 signals at different backgrounds and stages. Arrowheads indicate bivalent stretches lacking SYCP3 signals in KO. (I) Proportion statistics of spermatocytes with discontinuous SYCP3 signals. At least 200 cells of each mouse were counted. n=3 for WT and n=4 for KO. (J) Meiotic stage frequencies in (D-H). At least 200 cells of each mouse were counted. n=3.


( C ) Nuclear - spread spermatocytes from WT and KO mice were immunostained for SYCP3 and MLH1 . Representative images of pachynema are shown . Magnified views are indicated by dashed areas . ( D ) Number of bivalents without an MLH1 focus per pachytene spermatocyte in ( C ) . n = 3 mice . Nor , normal pachynema ; L 1 , pachynema - like 1 ; L 2 , pachynema - like 2 .

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(C) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 and MLH1. Representative images of pachynema are shown. Magnified views are indicated by dashed areas. (D) Number of bivalents without an MLH1 focus per pachytene spermatocyte in (C). n=3 mice. Nor, normal pachynema; L 1, pachynema-like 1; L 2, pachynema-like 2.


( E - H ) Nuclear - spread spermatocytes from WT and KO mice were immunostained for SYCP3 together with RAD51 ( E ) or DMC1 ( G ) and quantification of RAD51 ( F ) or DMC1 ( H ) focus numbers at early zygonema , late zygonema , and pachynema . Representative images of early zygonema are shown . n = 3 mice .

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(E-H) Nuclear-spread spermatocytes from WT and KO mice were immunostained for SYCP3 together with RAD51 (E) or DMC1 (G) and quantification of RAD51 (F) or DMC1 (H) focus numbers at early zygonema, late zygonema, and pachynema. Representative images of early zygonema are shown. n=3 mice.


( A - D ) Representative nuclear spreads of WT and KO spermatocytes at leptonema ( A ) , zygonema ( B ) , pachynema ( C ) and diplonema ( D ) . Spermatocytes were immunostained for REC8 and SYCP3 . REC8 foci distribute along SYCP3 - labeled structures . Magnified views are indicated by dashed areas .

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(A-D) Representative nuclear spreads of WT and KO spermatocytes at leptonema (A), zygonema (B), pachynema (C) and diplonema (D). Spermatocytes were immunostained for REC8 and SYCP3. REC8 foci distribute along SYCP3-labeled structures. Magnified views are indicated by dashed areas.


( E ) Inter - REC8 distances along axial elements . Five stretches or homologs were measured and the average distance was calculated for each nucleus . Each point represents one nucleus . The upper panel is the schematic representation for distance measurement . n = 3 mice .

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(E) Inter-REC8 distances along axial elements. Five stretches or homologs were measured and the average distance was calculated for each nucleus. Each point represents one nucleus. The upper panel is the schematic representation for distance measurement. n=3 mice.


( F ) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining . n = 3 mice . ( G ) Normalized density of REC8 in ( F ) . n = 3 mice .

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(F) Western blot analyses for REC8 and SEPARASE in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (G) Normalized density of REC8 in (F). n=3 mice.


( E ) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining . n = 3 mice . ( F ) Normalized density of SECURIN and CYCLIN B1 in ( E ) . n = 3 mice .

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(E) Western blot analyses for SECURIN and CYCLIN B1 in spermatocytes isolated by FACS after Hoechst staining. n=3 mice. (F) Normalized density of SECURIN and CYCLIN B1 in (E). n=3 mice.


( F ) Western blot analyses for REC8 and SEPARASE for cells in ( E ) . n = 3 biological replicates . ( G ) Normalized density of REC8 in ( F ) . n = 3 biological replicates .

(F) Western blot analyses for REC8 and SEPARASE for cells in (E). n=3 biological replicates. (G) Normalized density of REC8 in (F). n=3 biological replicates.


( H ) Western blot analyses for SECURIN and CYCLIN B1 for cells in ( E ) . n = 3 biological replicates . ( I ) Normalized density of SECURIN and CYCLIN B1 in ( H ) . n = 3 biological replicates .

(H) Western blot analyses for SECURIN and CYCLIN B1 for cells in (E). n=3 biological replicates. (I) Normalized density of SECURIN and CYCLIN B1 in (H). n=3 biological replicates.


( C ) End fusion analysis for domain deletion mutants of Rap1 . NotI - digested genomic DNA from nitrogen - starved cells was analyzed by pulsed - field gel electrophoresis ( PFGE ) and Southern blot with probes specific for the terminal C , I , L , and M fragments from chromosomes I and II .

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(C) End fusion analysis for domain deletion mutants of Rap1. NotI-digested genomic DNA from nitrogen-starved cells was analyzed by pulsed-field gel electrophoresis (PFGE) and Southern blot with probes specific for the terminal C, I, L, and M fragments from chromosomes I and II.


( D ) PFGE analysis as in ( C ) for nitrogen - starved cells harboring N - terminal truncation mutants of Rap1 . All mutants were V5 - tagged at their C - terminus .

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(D) PFGE analysis as in (C) for nitrogen-starved cells harboring N-terminal truncation mutants of Rap1. All mutants were V5-tagged at their C-terminus.


( A ) Telomere length analysis of cells from different sequential restreaks following introduction of Poz1 - V5 - Rap1 _ 491 - 693 into rap1 ∆ cells . Telomere length was assessed by Southern blotting of EcoRI - digested genomic DNA probed with a telomere specific probe . ( B ) PFGE analysis of nitrogen - starved Poz1 - V5 - Rap1 _ 491 - 693 cells after 5 and 14 restreaks .

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(A) Telomere length analysis of cells from different sequential restreaks following introduction of Poz1-V5-Rap1_491-693 into rap1∆ cells. Telomere length was assessed by Southern blotting of EcoRI-digested genomic DNA probed with a telomere specific probe. (B) PFGE analysis of nitrogen-starved Poz1-V5-Rap1_491-693 cells after 5 and 14 restreaks.


( C ) Telomere length analysis of cells with Rap1 ∆ RCT fused to Taz1 or Poz1 with intervening V5 tag . The fusion protein was integrated at the endogenous rap1 locus in the context of taz1 ∆ or poz1 ∆ , respectively . Different sequential restreaks following introduction of the fusion construct were analyzed . ( D ) PFGE analysis for nitrogen - starved Rap1 ∆ RCT - V5 - Taz1 and Rap1 ∆ RCT - V5 - Poz1 from 2nd restreak .

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(C) Telomere length analysis of cells with Rap1∆RCT fused to Taz1 or Poz1 with intervening V5 tag. The fusion protein was integrated at the endogenous rap1 locus in the context of taz1∆ or poz1∆, respectively. Different sequential restreaks following introduction of the fusion construct were analyzed. (D) PFGE analysis for nitrogen-starved Rap1∆RCT-V5-Taz1 and Rap1∆RCT-V5-Poz1 from 2nd restreak.


( B ) PFGE analysis of cells containing plasmids of Rap1 _ 440 - 693 with mutations of the amino acids identified in ( A ) in the context of rap1 ∆ . Cells were subjected to nitrogen - starvation after recovery from transformation ( Restreak 0 ) . ( C ) PFGE analysis of nitrogen - starved cells harboring the same mutations after 5 restreaks on plates ( approximately 110 generations ) . ( D ) Quantification of ( B ) and ( C ) using Fiji . The y - axis shows the percentage of intensity represented by the three fusion bands ( I + L , I + M and L + M ) relative to the total intensity of the fused and unfused signals . WT : wildtype Rap1 _ 440 - 693 .

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(B) PFGE analysis of cells containing plasmids of Rap1_440-693 with mutations of the amino acids identified in (A) in the context of rap1∆. Cells were subjected to nitrogen-starvation after recovery from transformation (Restreak 0). (C) PFGE analysis of nitrogen-starved cells harboring the same mutations after 5 restreaks on plates (approximately 110 generations). (D) Quantification of (B) and (C) using Fiji. The y-axis shows the percentage of intensity represented by the three fusion bands (I+L, I+M and L+M) relative to the total intensity of the fused and unfused signals. WT: wildtype Rap1_440-693.


( B ) Western blot analysis of N - terminally V5 - tagged truncation mutants using anti - V5 antibody . Anti - α - tubulin was used as loading control . Quantification of Rap1 mutant protein levels relative to WT normalized to α - tubulin is shown beneath the gel .

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(B) Western blot analysis of N-terminally V5-tagged truncation mutants using anti-V5 antibody. Anti-α-tubulin was used as loading control. Quantification of Rap1 mutant protein levels relative to WT normalized to α-tubulin is shown beneath the gel.


( C ) Telomere length analysis of the mutants by Southern blot with telomere specific probe . All mutants are V5 - tagged except for ∆ 491 - 638 untagged ( lane 7 ) .

(C) Telomere length analysis of the mutants by Southern blot with telomere specific probe. All mutants are V5-tagged except for ∆491-638 untagged (lane 7).


( D ) PFGE analysis of the same strains in ( C ) that were nitrogen - starved . ( E ) PFGE analysis of nitrogen - starved internal deletion mutants in poz1 ∆ background .

(D) PFGE analysis of the same strains in (C) that were nitrogen-starved. (E) PFGE analysis of nitrogen-starved internal deletion mutants in poz1∆ background.


( A ) PFGE analysis of nitrogen - starved N - terminally V5 - tagged full length Rap1 mutants . All mutants were integrated at the endogenous rap1 locus . M5A is the mutation of all 5 non - alanine amino acids under selection in the p - patch to alanine . ( B ) PFGE analysis of nitrogen - starved N - terminally V5 - tagged rap1 _ 440 - 693 mutants integrated at the endogenous rap1 locus .

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(A) PFGE analysis of nitrogen-starved N-terminally V5-tagged full length Rap1 mutants. All mutants were integrated at the endogenous rap1 locus. M5A is the mutation of all 5 non-alanine amino acids under selection in the p-patch to alanine. (B) PFGE analysis of nitrogen-starved N-terminally V5-tagged rap1_440-693 mutants integrated at the endogenous rap1 locus.


( C ) PFGE analysis of nitrogen - starved rap1 mutants in poz1 ∆ background .

(C) PFGE analysis of nitrogen-starved rap1 mutants in poz1∆ background.


( D ) PFGE analysis of nitrogen - starved rap1 mutants in ppm1 ∆ background . Untagged Rap1 are in lanes 1 - 2 and N - terminally V5 - tagged in lanes 3 - 7 .

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(D) PFGE analysis of nitrogen-starved rap1 mutants in ppm1∆ background. Untagged Rap1 are in lanes 1-2 and N-terminally V5-tagged in lanes 3-7.