[ a ] Representative of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT Xrcc1Nes - Cre mouse pups , immunostained for ADP - ribose ( red ) , NeuN to identify neurons ( green ) , counterstained with DAPI ( blue ) . were pretreated with PARP inhibitor ( 10 µM ) or vehicle for 2 h prior to fixation , with PARG inhibitor ( 10 μM ) additionally present for final hour . Scale bar 10 µm . [ b ] Histogram of mean ( ± ) relative pan - ADP - ribose in NeuN - positive hippocampal neurons pretreated with PARP inhibitor ( 5 µM ) or vehicle for 5 h prior to fixation , with PARG inhibitor ( 10 μM ) additionally present for final hour . Neurons were cultured from WT ( n = 6 mice , > 180 per condition ) , Xrcc1Nes - Cre ( n = 6 , > 180 ) , Parp1 + / - / Xrcc1Nes - Cre ( n = 3 , > 90 ) , Parp1 - / - / Xrcc1Nes - Cre ( n = 3 , > 90 ) . * indicates significant differences from WT ( Kruskal - Wallis ANOVA , p = 0 . 0013 Dunn ' s post - hoc ) .

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[a] Representative images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes-Cre mouse pups, immunostained for ADP-ribose (red), NeuN to identify neurons (green), and counterstained with DAPI (blue). Cells were pretreated with PARP inhibitor (10 µM) or vehicle for 2 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Scale bar 10 µm. [b] Histogram of mean (± SEM) relative pan-ADP-ribose fluorescence in NeuN-positive hippocampal neurons pretreated with PARP inhibitor (5 µM) or vehicle for 5 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Neurons were cultured from WT (n = 6 mice, > 180 cells per condition), Xrcc1Nes-Cre (n = 6, > 180), Parp1+/-/Xrcc1Nes-Cre (n = 3, > 90), and Parp1-/-/Xrcc1Nes-Cre (n = 3, > 90). * indicates significant differences from WT (Kruskal-Wallis ANOVA, p = 0.0013 and Dunn's post-hoc tests).


Summary histogram of mean ( ± ) of integrated responses for synapses for each condition . Synapse number is same as in ( D - F ) . Responses are significantly higher in Xrcc1Nes - Cre synapses versus both WT Parp1 - / - / Xrcc1Nes - Cre ( one - way ANOVA , * p < 0 . 0033 , pairwise student ' s t - ) .

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Summary histogram of the mean (± SEM) of the integrated fluorescence responses for all individual synapses for each condition. Synapse number is the same as in (D-F). Responses are significantly higher in Xrcc1Nes-Cre synapses versus both WT and Parp1-/-/Xrcc1Nes-Cre (one-way ANOVA, *p < 0.0033, and pairwise student's t-tests).


A . survival of - sex - matched HDAC6 + / + mice HDAC6 - / - mice was monitored for 9 days after intravenous VSV - Indiana infection ( 2 × 108 pfu / mouse ; n = 12 per group ; log - rank ) .

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A. The survival of age- and sex-matched HDAC6+/+ mice and HDAC6-/- mice was monitored for 9 days after intravenous VSV-Indiana infection (2 × 108 pfu/mouse; n = 12 per group; log-rank test).


B . Determination of viral load in organs by standard plaque . HDAC6 + / + HDAC6 - / - mice were intravenously infected with VSV - Indiana , brain spleen were collected at 5 dpi ( 2 × 108 pfu / mouse ; n = 6 per group ; Mann - Whitney ) .

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B. Determination of the viral load in organs by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-Indiana, and the brain and spleen were collected at 5 dpi (2 × 108 pfu/mouse; n = 6 per group; Mann-Whitney test).


C . Determination of viral loads in organs by Q - PCR of VSV viral transcripts . brain spleen were collected at 5 dpi after HDAC6 + / + HDAC6 - / - mice were intravenously infected with VSV - Indiana ( 2 × 108 pfu / mouse ; n = 9 per group ; Mann - Whitney ) .

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C. Determination of viral loads in organs by Q-PCR of VSV viral transcripts. The brain and spleen were collected at 5 dpi after HDAC6+/+and HDAC6-/- mice were intravenously infected with VSV-Indiana (2 × 108 pfu/mouse; n = 9 per group; Mann-Whitney test).


D . viral load in serum was assessed by standard plaque . HDAC6 + / + HDAC6 - / - mice were intravenously infected with VSV - GFP . Serum was collected at indicated time points analyzed ( 4 × 108 pfu / mouse ; n = 3 per group ; Student ' s t - ) .

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D. The viral load in the serum was assessed by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-GFP. Serum was collected at the indicated time points and analyzed (4 × 108 pfu/mouse; n = 3 per group; Student's t-test).


E . ELISA of IFN - β ( left ) IL - 6 ( right ) in serum of mice described in ( d ) ( Student ' s t - ) .

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E. ELISA of IFN-β (left) and IL-6 (right) in the serum of mice described in (d) (Student's t-test).


F . ELISA of serum IFN - β ( left ) IL - 6 ( right ) after poly ( I : C ) injection . HDAC6 + / + HDAC6 - / - mice were intravenously injected with poly ( I : C ) . Serum was collected at indicated time points analyzed ( 200 μg / mouse ; n = 3 per group ; Student ' s t - ) .

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F. ELISA of serum IFN-β (left) and IL-6 (right) after poly(I:C) injection. HDAC6+/+ and HDAC6-/- mice were intravenously injected with poly(I:C). Serum was collected at the indicated time points and analyzed (200 μg/mouse; n = 3 per group; Student's t-test).


A , B . replication at 12 24 hpi , in HDAC6 + / + HDAC6 - / - BMDMs in response to VSV - GFP ( MOI = 10 ) infection ( A ) PR8 - GFP ( MOI = 5 ) infection ( B ) .

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A, B. virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- BMDMs in response to VSV-GFP (MOI = 10) infection (A) and PR8-GFP (MOI = 5) infection (B).


C . replication in HDAC6 + / + HDAC6 - / - BMDMs in response to HSV - GFP ( MOI = 2 ) infection .

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C. Virus replication in HDAC6+/+ and HDAC6-/- BMDMs in response to HSV-GFP (MOI = 2) infection.


D . replication in HDAC6 + / + HDAC6 - / - PBMCs in response to VSV - GFP ( MOI = 10 ) infection .

D. Virus replication in HDAC6+/+ and HDAC6-/- PBMCs in response to VSV-GFP (MOI = 10) infection.


E . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) in supernatant of ( A ) ( B ) , in HDAC6 + / + HDAC6 - / - BMDMs treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) .

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E. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in HDAC6+/+ and HDAC6-/- BMDMs treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).


F . Immunoblot of phosphorylated inactive forms of IRF3 , IKBα , TBK1 , RIG - I , MAVS , HDAC6 , β - actin at indicated times ( 0 , 2 , 4 , 8 , 16 h ) in HDAC6 + / + HDAC6 - / - BMDMs . BMDMs were stimulated with PR8 - GFP ( MOI = 3 ) .

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F. Immunoblot analysis of the phosphorylated and inactive forms of IRF3, IKBα, TBK1, RIG-I, MAVS, HDAC6, and β-actin at the indicated times (0, 2, 4, 8, and 16 h) in HDAC6+/+ and HDAC6-/- BMDMs. BMDMs were stimulated with PR8-GFP (MOI = 3).


G . Induction of mRNA for type I IFN , IL - 6 , other IFN - related antiviral genes in HDAC6 + / + HDAC6 - / - BMDMs in response to a RIG - I agonist stimulation at 6 h . HDAC6 + / + HDAC6 - / - BMDMs were stimulated with 5 ' ppp - dsRNA ( 0 . 5 µg / ml ) for 6 h .

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G. Induction of mRNA for type I IFN, IL-6, and other IFN-related antiviral genes in HDAC6+/+ and HDAC6-/- BMDMs in response to a RIG-I agonist stimulation at 6 h. HDAC6+/+ and HDAC6-/- BMDMs were stimulated with 5'ppp-dsRNA (0.5 µg/ml) for 6 h.


A , B . microscopy at 24 hpi showing green absorbance at 12 24 hpi , replication at 12 24 hpi , in control HDAC6 knockdown RAW264 . 7 in response to VSV - GFP ( MOI = 1 ) infection ( A ) PR8 - GFP ( MOI = 1 ) infection ( B ) . bar ,

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A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in control and HDAC6 knockdown RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm


C . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) in supernatant of ( A ) ( B ) , in control HDAC6 knockdown RAW264 . 7 treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) .

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C. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in control and HDAC6 knockdown RAW264.7 cells treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).


D , E . microscopy at 24 hpi showing green absorbance at 12 24 hpi , replication at 12 24 hpi , in HDAC6 + / + HDAC6 - / - MEFs in response to VSV - GFP ( MOI = 1 ) infection ( D ) PR8 - GFP ( MOI = 1 ) infection ( E ) . bar ,

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D, E. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- MEFs in response to VSV-GFP (MOI = 1) infection (D) and PR8-GFP (MOI = 1) infection (E). bar, 50µm


F . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) in supernatant of ( D ) ( E ) , in HDAC6 + / + HDAC6 - / - MEFs transfected with poly ( I : C ) ( 1 µg / ml ) .

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F. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (D) and (E), and in HDAC6+/+ and HDAC6-/- MEFs transfected with poly(I:C) (1 µg/ml).


A , B . microscopy at 24 hpi showing green absorbance at 12 24 hpi , replication at 12 24 hpi , in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 in response to VSV - GFP ( MOI = 1 ) infection ( A ) PR8 - GFP ( MOI = 1 ) infection ( B ) . bar , 100µm .

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A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm.


C . ELISA of IFN - b ( upper ) IL - 6 ( lower ) in supernatant of ( A ) , ( B ) , in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 treated with poly ( I : C ) ( 20 μg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) .

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C. ELISA of IFN-b (upper) and IL-6 (lower) levels in the supernatant of (A), (B), and in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).


D , E . Luciferase in 293T transfected with an IFN - β luciferase promoter TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) , followed by PR8 - GFP ( MOI = 2 ) infection ( D ) or by poly ( I : C ) ( E ) ( 1 μg / ml ) transfection for another 12 h .

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D, E. Luciferase assay in 293T cells transfected with an IFN-β luciferase promoter and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng), followed by PR8-GFP (MOI = 2) infection (D) or by poly(I:C) (E) (1 μg/ml) transfection for another 12 h.


F , G . Luciferase in 293T transfected with RIG - I ( F ) , MDA - 5 ( G ) , an IFN - β luciferase promoter , TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) . 24 h later , IFN - β activity was measured by luciferase reporter .

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F, G. Luciferase assay in 293T cells transfected with RIG-I (F), MDA-5 (G), an IFN-β luciferase promoter, and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng). 24 h later, IFN-β activity was measured by luciferase reporter assay.


A . 293T were transfected with poly ( I : C ) lysates were prepared at indicated times for co - immunoprecipitation of endogenous HDAC6 RIG - I . Actin was used as loading control .

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A. 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control.


B . BMDMs were isolated from HDAC6 + / + HDAC6 - / - mice were transfected with 5ˊppp - dsRNA . Whole lysates were prepared after 8 h of transfection co - immunoprecipitation of RIG - I HDAC6 was performed .

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B. BMDMs were isolated from HDAC6+/+ and HDAC6-/- mice and were transfected with 5ˊppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed.


C . HeLa were transfected with 5ˊppp - dsRNA . were stained with anti - RIG - I ( red ) anti - HDAC6 H300 ( green ) antibodies after 8 h of transfection . Nuclei were stained with DAPI ( blue ) ( bar , 20µm ) .

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C. HeLa cells were transfected with 5ˊppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (bar, 20µm).


D . microscopy , green absorbance replication at 24hpi in RIG - I + / + RIG - I - / - MEFs . were transfected with si - HDAC6 si - control for 36 h , followed infection with VSV - GFP ( MOI = 1 ) . bar , 100µm .

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D. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24hpi in RIG-I+/+ and RIG-I-/- MEFs. Cells were transfected with si-HDAC6 and si-control for 36 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm.


E . HDAC6 + / + , HDAC6 - / - MEFs , HDAC6 - / - MEFs reconstituted with HDAC6 wild - type were subjected to immunoprecipitation using an anti - RIG - I antibody immunoblotted with anti - acetyl - lysine anti - RIG - I antibodies . Acetyl - tubulin was measured in total lysates to determine HDAC6 deacetylase activity .

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E. HDAC6+/+, HDAC6-/- MEFs, and HDAC6-/- MEFs reconstituted with HDAC6 wild-type were subjected to immunoprecipitation using an anti-RIG-I antibody and immunoblotted with anti-acetyl-lysine and anti-RIG-I antibodies. Acetyl-tubulin was measured in total lysates to determine HDAC6 deacetylase activity.


F . Whole lysates from HDAC6 + / + HDAC6 - / - MEFs were prepared used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I , anti - HDAC6 , anti - β - actin antibodies . of pull - downed RIG - I was quantified .

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F. Whole cell lysates from HDAC6+/+ and HDAC6-/- MEFs were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I, anti-HDAC6, and anti-β-actin antibodies. Intensity of pull-downed RIG-I was quantified.


G . Whole lysates from HDAC6 - / - MEFs HDAC6 - / - MEFs reconstituted with HDAC6 wild - type HDAC6 - CDM were prepared used in dsRNA pull - down assays . Samples were analyzed by western blotting with anti - RIG - I anti - HDAC6 antibodies . of pull - downed RIG - I was quantified .

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G. Whole cell lysates from HDAC6-/- MEFs and HDAC6-/- MEFs reconstituted with HDAC6 wild-type and HDAC6-CDM were prepared and used in dsRNA pull-down assays. Samples were analyzed by western blotting with anti-RIG-I and anti-HDAC6 antibodies. Intensity of pull-downed RIG-I was quantified.


A , B . 293T were transfected with indicated expression plasmids . At 1 day post - transfection , whole lysates were subjected to dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with an anti - FLAG antibody .

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A, B. 293T cells were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were subjected to dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with an anti-FLAG antibody.


C . A luciferase was performed using 293T transfected with IFN - βluciferase promoter TK - Renilla together with 2 ng of RIG - I wild - type , RIG - I 858Q , RIG - I 858R , RIG - I K909Q , or RIG - I K909R mutant , followed by transfection with poly ( I : C ) ( 1 μg / ml ) for another 12 h .

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

C. A luciferase assay was performed using 293T cells transfected with the IFN-βluciferase promoter and TK-Renilla together with 2 ng of RIG-I wild-type, RIG-I 858Q, RIG-I 858R, RIG-I K909Q, or RIG-I K909R mutant, followed by transfection with poly(I:C) (1 μg/ml) for another 12 h.


D . microscopy , green absorbance , replication at 24 hpi in RIG - I - / - MEFs . were transiently transfected with empty vector , RIG - I wild - type , RIG - I K909Q , or RIG - I K909R mutant for 24 h , followed infection with VSV - GFP ( MOI = 1 ) . bar ,

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

D. Fluorescence microscopy, green fluorescence absorbance, and virus replication at 24 hpi in RIG-I-/- MEFs. Cells were transiently transfected with empty vector, RIG-I wild-type, RIG-I K909Q, or RIG-I K909R mutant for 24 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm


E . HDAC6 - / - MEFs were transfected with indicated expression plasmids . At 1 day post - transfection , whole lysates were prepared used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I anti - β - actin antibodies .

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E. HDAC6-/- MEFs were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I and anti-β-actin antibodies.


F . 293T were transfected with 2 μg of FLAG - tagged RIG - I 1 , 5 , or 10 μg of V5 - tagged HDAC6 . At 36 h post - transfection , whole lysates were prepared for co - immunoprecipitation of V5 - tagged HDAC6 acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody .

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F. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I and 1, 5, or 10 μg of V5-tagged HDAC6. At 36 h post-transfection, whole cell lysates were prepared for co-immunoprecipitation analysis of V5-tagged HDAC6 and acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody.


G . 293T were transfected with 2 μg of FLAG - tagged RIG - I . At 24 h post - transfection , were infected with VSV - GFP ( MOI = 0 . 1 ) . At 2 4 h post - infection , whole lysates were prepared for immunoprecipitation of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . of ac - 909K normalized to Flag - RIG - I was quantified .

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G. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I. At 24 h post-transfection, cells were infected with VSV-GFP (MOI = 0.1). At 2 and 4 h post-infection, whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to Flag-RIG-I was quantified.


H . whole lysates were prepared for immunoprecipitation of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . of ac - 909K normalized to RIG - I was quantified .

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

H. whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to RIG-I was quantified.


A . structural modeling result of RIG - I / dsRNA positions of lysine residue were presented . RIG - I helicase A B are shown as green pale blue ribbons . dsRNA strands were presented as purple yellow rods , respectively . Two lysine residues , K907 K909 , on RIG - I that interact with dsRNA are indicated as violet sticks . K907 forms hydrogen bonding with neither 5 ´ - ppp dsRNA nor K907 . B , C . Acetylated K909 K907 on RIG - I are indicated as green sticks . There were hydrogen bonding between NH2 of K907 oxygen of acetyl - K909 .

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A. The structural modeling result of RIG-I/dsRNA complex and positions of the lysine residue were presented. RIG-I helicase A and B are shown as green and pale blue ribbons. The dsRNA strands were presented as purple and yellow rods, respectively. Two lysine residues, K907 and K909, on RIG-I that interact with dsRNA are indicated as violet sticks. K907 forms hydrogen bonding with neither 5´-ppp dsRNA nor K907.B, C. Acetylated K909 and K907 on RIG-I are indicated as green sticks. There were hydrogen bonding between NH2 of K907 and oxygen of acetyl-K909.


( A ) Intracellular endogenous Aβ42 ( green ) , Ankyrin - G ( AnkG ; magenta ) GFP ( blue ) in siBin1 - , siCD2AP - siControl - treated cortical neurons ( neurons ) expressing GFP immunolabelled at 9 DIV with anti - Aβ42 ( clone 12F4 ) anti - AnkG , analysed by spinning - disk confocal microscopy . rectangles indicate dendrites ( Dd ) axons ( Ax ) magnified below showing Aβ42 in axons dendrites outlined on AnkG ( magenta ) GFP ( blue ) , respectively . Scale bars , 10 µm . ( B ) Aβ42 profiles in dendrites ( Dd ) of siControl ( grey ) , siBin1 ( blue ) siCD2AP ( red ) neurons shown in ( c ) . ( C ) Aβ42 profiles in axons ( Ax ) of siControl ( grey ) , siBin1 ( blue ) siCD2AP ( red ) neurons shown in ( c ) .

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(A) Intracellular endogenous Aβ42 (green), Ankyrin-G (AnkG; magenta) and GFP (blue) in siBin1- , siCD2AP- and siControl-treated primary cortical neurons (neurons) expressing GFP immunolabelled at 9 DIV with anti-Aβ42 (clone 12F4) and anti-AnkG, analysed by spinning-disk confocal microscopy. The white rectangles indicate the dendrites (Dd) and axons (Ax) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG (magenta) and GFP (blue), respectively. Scale bars, 10 µm.(B) Aβ42 line profiles in dendrites (Dd) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).(C) Aβ42 line profiles in axons (Ax) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).


( D ) of Aβ42 ( 12F4 ) in ( CB ) , dendrite ( Dd ) axon ( Ax ) ( n = 5 , NCB = 44 - 53 , NDd = 90 - 120 , NAx = 60 - 74 ; * * * * PCB < 0 . 0001 siBin1 vs . siControl , * * * PCB < 0 . 001 siCD2AP vs . siControl , * * * * PDd < 0 . 0001 siBin1 vs . siControl , * * * * PDd < 0 . 0001 siCD2AP vs . siControl , * * * * PAx < 0 . 0001 siBin1 vs . siControl , t - , mean ± ) .

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(D) Quantification of Aβ42 (12F4) intensity in cell body (CB), dendrite (Dd) and axon (Ax) (n=5, NCB=44-53, NDd=90-120, NAx=60-74; ****PCB<0.0001 siBin1 vs. siControl, ***PCB<0.001 siCD2AP vs. siControl, ****PDd<0.0001 siBin1 vs. siControl, ****PDd<0.0001 siCD2AP vs. siControl, ****PAx<0.0001 siBin1 vs. siControl, t-test, mean ± SEM).


( E ) of extracellular endogenous Aβ40 , Aβ42 of Aβ42 / Aβ40 by ELISA of conditioned media of 9 DIV siBin1 , siCD2AP or siControl neurons ( n = 6 ; * PAβ40 = 0 . 0270 siBin1 vs . siControl , * PAβ42 / 40 = 0 . 0378 siBin1 vs . siControl , * PAβ42 / 40 = 0 . 0463 siCD2AP vs . siControl , t - , mean ± ) .

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(E) Quantification of extracellular endogenous Aβ40, Aβ42 and of Aβ42/Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1, siCD2AP or siControl neurons (n=6; *PAβ40=0.0270 siBin1 vs. siControl, *PAβ42/40=0.0378 siBin1 vs. siControl, *PAβ42/40=0.0463 siCD2AP vs. siControl, t-test, mean ± SEM).


( F ) Endogenous APP APP - CTFs by western blot with anti - APP antibody ( Y188 ) of siBin1 - , siCD2AP - or siControl - treated neurons at 9 DIV . ( G ) of APP APP - CTFs normalized to APP ( n = 4 ; * PAPP = 0 . 0355 siBin1 vs . siControl , * * * PAPPCTFs / APP < 0 . 001 siBin1 vs . siControl , * * * * PAPPCTFs / APP < 0 . 0001 siCD2AP vs . siControl , t - , mean ± ) .

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(F) Endogenous APP and APP-CTFs levels by western blot with anti-APP antibody (Y188) of siBin1-, siCD2AP- or siControl-treated neurons at 9 DIV.(G) Quantification of APP and APP-CTFs levels normalized to APP (n=4; *PAPP=0.0355 siBin1 vs. siControl, ***PAPPCTFs/APP<0.001 siBin1 vs. siControl, ****PAPPCTFs/APP<0.0001 siCD2AP vs. siControl, t-test, mean ± SEM).


N2a treated with siBin1 , siCD2AP or siControl . ( B ) Endocytosed APP detected after a 10 min with 22C11 APP - RFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( C ) amount of endocytosed APP at 10 min was quantified normalized to APP - RFP ( n = 3 , NsiControl = 56 , NsiBin1 = 70 , NsiCD2AP = 42 ; mean ± ) . ( D ) Non - degraded APP detected after 10 min 60 min chase with 22C11 APP - RFP ( insets ) , analysed by epifluorescence microscopy . are outlined in . Scale bars , 10 µm . ( E ) APP degradation was assessed by decrease in amount of endocytosed APP at 60 min relative to time 0 ( 10 min ) in siControl normalized to APP - RFP ( n60min = 3 , NsiControl = 68 , NsiBin1 = 43 , NsiCD2AP = 64 ; * * * * PAPP60min < 0 . 0001 , t - , mean ± ) .

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

N2a cells treated with siBin1, siCD2AP or siControl.(B) Endocytosed APP detected after a 10 min pulse with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(C) The amount of endocytosed APP fluorescence at 10 min was quantified and normalized to APP-RFP fluorescence (n=3, NsiControl=56, NsiBin1=70, NsiCD2AP =42; mean ± SEM).(D) Non-degraded APP detected after 10 min pulse and 60 min chase with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Cells are outlined in white. Scale bars, 10 µm.(E) APP degradation was assessed by the decrease in the amount of endocytosed APP fluorescence at 60 min relative to time 0 (10 min pulse) in siControl cells normalized to APP-RFP fluorescence (n60min=3, NsiControl=68, NsiBin1=43, NsiCD2AP=64; ****PAPP60min<0.0001, t-test, mean ± SEM).


N2a treated with siBin1 , siCD2AP or siControl . ( G ) Endocytosed BACE1 detected upon 5 min with M1 BACE1 - GFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( H ) amount of endocytosed BACE1 per was quantified as of siControl normalized to BACE1 - GFP ( n = 3 , NsiControl = 86 , NsiBin1 = 72 , NsiCD2AP = 113 ; mean ± ) . ( I ) Recycled BACE1 detected at plasma of non - permeabilized with M1 , upon a 10 min , acid stripping 20 min chase , BACE1 - GFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( J ) amount of recycled BACE1 was quantified as in ( h ) ( n = 3 , NsiControl = 94 , NsiBin1 = 58 , NsiCD2AP = 109 ; * * * * P < 0 . 0001 siBin1 vs . siControl , t - , mean ± ) . ( K ) Non - recycled BACE1 detected in acid - stripped permeabilized - chased as in ( i ) with M1 BACE1 - GFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( L ) amount of non - recycled BACE1 was quantified as in ( h ) ( n = 3 , NsiControl = 61 , NsiBin1 = 51 ; * * * * P < 0 . 0001 siBin1 vs . siControl , t - ; n = 2 , NsiCD2AP = 37 ; mean ± ) .

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

N2a cells treated with siBin1, siCD2AP or siControl.(G) Endocytosed BACE1 detected upon 5 min pulse with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(H) The amount of endocytosed BACE1 per cell was quantified as percentage of siControl normalized to BACE1-GFP (n=3, NsiControl=86, NsiBin1=72, NsiCD2AP=113; mean ± SEM).(I) Recycled BACE1 detected at the plasma membrane of non-permeabilized cells with M1, upon a 10 min pulse, acid stripping and 20 min chase, and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(J) The amount of recycled BACE1 was quantified as in (h) (n=3, NsiControl=94, NsiBin1=58, NsiCD2AP=109; ****P<0.0001 siBin1 vs. siControl, t-test, mean ± SEM).(K) Non-recycled BACE1 detected in acid-stripped permeabilized cells pulse-chased as in (i) with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(L) The amount of non-recycled BACE1 was quantified as in (h) (n=3, NsiControl=61, NsiBin1=51; ****P<0.0001 siBin1 vs. siControl, t-test; n=2, NsiCD2AP=37; mean ± SEM).


( A - D ) APP endocytic trafficking followed in neurons expressing APP - RFP treated with siCD2AP or siControl using a - chase with 22C11 , analysed by epifluorescence microscopy . ( A ) Endocytosed APP detected with 22C11 ( 10 min ; top panels ) APP - RFP ( bottom panels ) . Arrowheads identify axons ( Ax ) dendrites ( Dd ) magnified on right . Scale bars , 10 µm . ( B ) amount of endocytosed APP ( 10 min ) in , dendrites axons normalized to APP - RFP expression in quantified as of siControl ( n = 3 , NsiControl = 19 , NsiCD2AP = 20 ; mean ± ) . ( C ) Endocytosed APP detected with 22C11 ( 10 min , 60 min chase ; top panels ) APP - RFP ( bottom panels ) . Arrowheads identify axons ( Ax ) dendrites ( Dd ) magnified on right . Scale bars , 10 µm . ( D ) amount of endocytosed APP ( 60 min chase ) was quantified as in ( b ) ( n = 3 , NsiControl = 23 , NsiCD2AP = 19 ; * * PCB = 0 . 001 siCD2AP vs . siControl , * * * PDd = 0 . 0004 siCD2AP vs . siControl , t - ; # P = 0 . 0347 Dendrite vs . , # # # P = 0 . 0008 Dendrite vs . Axon , 1 - way ANOVA with Tukey ' s multiple comparisons ; mean ± ) .

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(A - D) APP endocytic trafficking followed in neurons expressing APP-RFP treated with siCD2AP or siControl using a pulse-chase assay with 22C11, analysed by epifluorescence microscopy.(A) Endocytosed APP detected with 22C11 (10 min pulse; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(B) The amount of endocytosed APP (10 min) in cell body, dendrites and axons normalized to APP-RFP expression in the cell body quantified as percentage of siControl (n=3, NsiControl=19, NsiCD2AP=20; mean ± SEM).(C) Endocytosed APP detected with 22C11 (10 min pulse, 60 min chase; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(D) The amount of endocytosed APP (60 min chase) was quantified as in (b) (n=3, NsiControl=23, NsiCD2AP=19; **PCB=0.001 siCD2AP vs. siControl, ***PDd=0.0004 siCD2AP vs. siControl, t-test; #P=0.0347 Dendrite vs. Cell body, ###P=0.0008 Dendrite vs. Axon, 1-way ANOVA with Tukey's multiple comparisons test; mean ± SEM).


( E ) Degradation of surface biotinylated APP ( biotin APP ) ( time 0 ) chased for 20 60 min in neurons treated with siCD2AP , siBin1 or siControl . Biotin APP total APP were detected with anti - APP ( Y188 ) by western blot . ( F ) of biotinylated APP normalized to at time 0 ( n = 3 - 4 , * P60min = 0 . 0198 , t - ; replicates mean ) .

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(E) Degradation of surface biotinylated APP (biotin APP) (time 0) chased for 20 and 60 min in neurons treated with siCD2AP, siBin1 or siControl. Biotin APP and total APP were detected with anti-APP (Y188) by western blot.(F) Quantification of biotinylated APP normalized to levels at time 0 (n=3-4, *P60min=0.0198, t-test; replicates and mean).


( G H ) CD2AP ( green ) APP ( magenta ) localisation in dendrites ( G ) axons ( I ) of neurons expressing APP - RFP immunolabelled at 11 DIV with anti - CD2AP , analysed by spinning - disk confocal microscopy . Scale bars , 10 µm . squares are magnified on right . Scale bars , 1 µm . ( I ) of colocalisation between CD2AP APP in axons dendrites ( n = 3 - 4 , NDd = 19 , NAx = 17 ; * * * * P < 0 . 0001 , t - , mean ± ) .

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(G and H) CD2AP (green) and APP (magenta) localisation in dendrites (G) and axons (I) of neurons expressing APP-RFP immunolabelled at 11 DIV with anti-CD2AP, analysed by spinning-disk confocal microscopy. Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(I) Quantification of colocalisation between CD2AP and APP in axons and dendrites (n=3-4, NDd=19, NAx=17; ****P<0.0001, t-test, mean ± SEM).


( J K ) CD2AP ( green ) Rab5 ( magenta ) localisation in dendrites ( J ) axons ( K ) of neurons expressing Rab5 - GFP analysed as in ( G H ) . Scale bars , 10 µm . squares are magnified on right . Scale bars , 1 µm . ( L ) of colocalisation between CD2AP Rab5 - positive endosomes in dendrites ( Dd ) axons ( Ax ) ( n = 3 , NDd = 17 , NAx = 23 , * * * * P < 0 . 0001 , t - , mean ± ) .

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

(J and K) CD2AP (green) and Rab5 (magenta) localisation in dendrites (J) and axons (K) of neurons expressing Rab5-GFP analysed as in (G and H). Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(L) Quantification of colocalisation between CD2AP and Rab5-positive endosomes in dendrites (Dd) and axons (Ax) (n=3, NDd=17, NAx=23, ****P<0.0001, t-test, mean ± SEM).


( A - D ) BACE1 endocytic trafficking followed in siBin1 - siControl - treated neurons expressing BACE1 - GFP N - terminally tagged with FLAG using a / chase with M1 , an anti - FLAG antibody , analysed by epifluorescence microscopy . ( A ) Endocytosed BACE1 detected with M1 ( 15 min ; top panels ) BACE1 - GFP ( bottom panels ) . Arrowheads identify axons ( Ax ) dendrites ( Dd ) magnified on right . Scale bars , 10 µm . ( B ) amount of endocytosed BACE1 per , dendrite axons was normalized to BACE1 - GFP expression in quantified as of siControl ( n = 3 , NsiControl = 17 , NsiBin1 = 19 , mean ± ) . ( C ) Recycled BACE1 detected with M1 ( 15 min , acid stripping 20 min chase ; top panels ) at plasma of non - permeabilized neurons BACE1 - GFP ( bottom panels ) . Arrowheads identify axons ( Ax ) dendrites ( Dd ) magnified on right . Scale bars , 10 µm . ( D ) amount of recycled BACE1 analysed as in ( b ) ( n = 4 , NsiControl = 31 , NsiBin1 = 38 , * * * PCB = 0 . 0002 * * * * PAx < 0 . 0001 siBin1 vs . siControl , t - ; # # P = 0 . 0014 Axon vs . Dendrite , 1 - way ANOVA with Tukey multiple comparisons ; mean ± ) .

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

(A - D) BACE1 endocytic trafficking followed in siBin1- and siControl-treated neurons expressing BACE1-GFP N-terminally tagged with FLAG using a pulse/chase assay with M1, an anti-FLAG antibody, analysed by epifluorescence microscopy.(A) Endocytosed BACE1 detected with M1 (15 min pulse; top panels) and BACE1-GFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(B) The amount of endocytosed BACE1 per cell body, dendrite and axons was normalized to BACE1-GFP expression in the cell body and quantified as percentage of siControl (n=3, NsiControl=17, NsiBin1=19, mean ± SEM).(C) Recycled BACE1 detected with M1 (15 min pulse, acid stripping and 20 min chase; top panels) at the plasma membrane of non-permeabilized neurons and BACE1-GFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(D) The amount of recycled BACE1 analysed as in (b) (n=4, NsiControl=31, NsiBin1=38, ***PCB=0.0002 and ****PAx<0.0001 siBin1 vs. siControl, t-test; ##P=0.0014 Axon vs. Dendrite, 1-way ANOVA with Tukey multiple comparisons test; mean ± SEM).


( E F ) Bin1 ( green ) BACE1 ( magenta ) localisation in axons ( E ) dendrites ( F ) of neurons expressing BACE1 - GFP immunolabelled at 11 DIV with anti - Bin1 , analysed by spinning - disk confocal microscopy . Scale bars , 10 µm . squares are magnified on right . Scale bars , 1 µm . ( G ) of colocalisation between Bin1 BACE1 in axons dendrites ( n = 3 , NDd = 17 , NAx = 21 ; * * * P = 0 . 0006 Axon vs . Dendrite , t - ; mean ± ) .

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

(E and F) Bin1 (green) and BACE1 (magenta) localisation in axons (E) and dendrites (F) of neurons expressing BACE1-GFP immunolabelled at 11 DIV with anti-Bin1, and analysed by spinning-disk confocal microscopy. Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(G) Quantification of colocalisation between Bin1 and BACE1 in axons and dendrites (n=3, NDd=17, NAx=21; ***P=0.0006 Axon vs. Dendrite, t-test; mean ± SEM).


( H I ) Bin1 ( green ) Rab5 ( magenta ) localisation in axons ( H ) dendrites ( I ) of neurons expressing Rab5 - GFP analysed as in ( E F ) . Scale bars , 10 µm . squares are magnified on right . Scale bars , 1 µm . ( J ) of colocalisation between Bin1 Rab5 - positive endosomes in axons ( Ax ) dendrites ( Dd ) ( n = 3 , NAx = 39 , NDd = 16 , * * * * P < 0 . 0001 , t - , mean ± ) .

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

(H and I) Bin1 (green) and Rab5 (magenta) localisation in axons (H) and dendrites (I) of neurons expressing Rab5-GFP analysed as in (E and F). Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(J) Quantification of colocalisation between Bin1 and Rab5-positive endosomes in axons (Ax) and dendrites (Dd) (n=3, NAx=39, NDd=16, ****P<0.0001, t-test, mean ± SEM).


( A ) Endocytosed APP ( green ) detected with 22C11 ( 10 min 60 min chase ) in EEA1 - positive early endosomes ( magenta ) in dendrites of siCD2AP - siControl - treated neurons expressing APP - RFP , analysed by spinning - disk confocal microscopy . Scale bar , 10 µm . squares indicate an EEA1 - positive endosome magnified on right . Scale bar , 1 µm . ( B ) of colocalisation between endocytosed APP EEA1 in dendrites ( n = 3 , NsiControl = 26 NsiCD2AP = 18 ; * * * * P < 0 . 0001 , t - ; mean ± ) .

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

(A) Endocytosed APP (green) detected with 22C11 (10 min pulse and 60 min chase) in EEA1-positive early endosomes (magenta) in dendrites of siCD2AP- and siControl-treated neurons expressing APP-RFP, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. The white squares indicate an EEA1-positive endosome magnified on the right. Scale bar, 1 µm.(B) Quantification of colocalisation between endocytosed APP and EEA1 in dendrites (n=3, NsiControl=26 NsiCD2AP=18; ****P<0.0001, t-test; mean ± SEM).


( C ) Non - recycled BACE1 ( green ) detected with M1 ( 15 min , acid stripping 20 min chase ) in Rab5 - positive early endosomes ( magenta ) in axons of siControl - siBin1 - treated neurons expressing Rab5 - mCherry BACE1 - GFP , analysed by spinning - disk confocal microscopy . Scale bar , 10 µm . squares indicate a Rab5 - positive endosome magnified on right . Scale bar , 1 µm . ( D ) of colocalisation between non - recycled BACE1 Rab5 in axons ( n = 3 , NsiControl = 19 , NsiBin1 = 24 ; * * * * P < 0 . 0001 , t - ; mean ± ) .

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

(C) Non-recycled BACE1 (green) detected with M1 (15 min pulse, acid stripping and 20 min chase) in Rab5-positive early endosomes (magenta) in axons of siControl- and siBin1-treated neurons expressing Rab5-mCherry and BACE1-GFP, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares indicate a Rab5-positive endosome magnified on the right. Scale bar, 1 µm.(D) Quantification of colocalisation between non-recycled BACE1 and Rab5 in axons (n=3, NsiControl=19, NsiBin1=24; ****P<0.0001, t-test; mean ± SEM).


( E ) APP ( magenta ) BACE1 ( green ) colocalisation in dendrites of siCD2AP - siControl - treated neurons expressing APP - RFP BACE1 - GFP upon DAPT treatment , recorded by time - lapse spinning - disk confocal microscopy for 120 sec ( 1 fps ) ( see supplementary movie 1 ) . APP BACE1 in dendrites at 0 sec are shown during 120 sec in kymographs ( bottom panels ) . Dotted lines in kymographs highlight APP vesicles positive for BACE1 . Scale bar , 10 µm . ( F ) of colocalisation between APP BACE1 in dendrites ( n = 3 , NsiControl = 18 , NsiCD2AP = 21 ; * * * * P < 0 . 0001 , t - ; mean ± )

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

(E) APP (magenta) and BACE1 (green) colocalisation in dendrites of siCD2AP- and siControl-treated neurons expressing APP-RFP and BACE1-GFP upon DAPT treatment, recorded by time-lapse spinning-disk confocal microscopy for 120 sec (1 fps) (see supplementary movie 1). APP and BACE1 in dendrites at 0 sec are shown and during 120 sec in kymographs (bottom panels). Dotted white lines in kymographs highlight APP vesicles positive for BACE1. Scale bar, 10 µm.(F) Quantification of the colocalisation between APP and BACE1 in dendrites (n=3, NsiControl=18, NsiCD2AP=21; ****P<0.0001, t-test; mean ± SEM)


( G ) APP ( magenta ) BACE1 ( green ) colocalisation in axons of siBin1 - siControl - treated neurons expressing APP - RFP BACE1 - GFP upon DAPT treatment , recorded by time - lapse spinning - disk confocal microscopy for 120 sec ( 1 fps ) ( see supplementary movie 2 ) . APP BACE1 in axons at 0 sec are shown during 120 sec in kymographs ( bottom panels ) . Dotted lines in kymographs highlight APP vesicles positive for BACE1 . Scale bar , 10 µm . ( H ) of colocalisation between APP BACE1 in axons ( n = 4 , NsiControl = 17 , NsiBin1 = 27 ; * * * * P < 0 . 0001 , t - ; mean ± ) .

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

(G) APP (magenta) and BACE1 (green) colocalisation in axons of siBin1- and siControl-treated neurons expressing APP-RFP and BACE1-GFP upon DAPT treatment, recorded by time-lapse spinning-disk confocal microscopy for 120 sec (1 fps) (see supplementary movie 2). APP and BACE1 in axons at 0 sec are shown and during 120 sec in kymographs (bottom panels). Dotted white lines in kymographs highlight APP vesicles positive for BACE1. Scale bar, 10 µm.(H) Quantification of the colocalisation between APP and BACE1 in axons (n=4, NsiControl =17, NsiBin1=27; ****P<0.0001, t-test; mean ± SEM).


( A ) BACE1 carriers in axons of siBin1 - or siControl - treated neurons expressing BACE1 - GFP , analysed by epifluorescence microscopy . Scale bar , 10 µm . rectangles indicate a BACE1 - positive carrier magnified on right . Scale bar , 1 µm . ( B ) of of siBin1 - or siControl - treated neurons displaying extended BACE1 carriers ( as shown in ( a ) ) ; ( n = 3 , NsiControl = 67 , NsiBin1 = 68 ; * P = 0 . 0199 , t - , mean ± ) . ( C ) of circularity of BACE1 carriers in axons in dendrites of siBin1 - or siControl - treated neurons . of BACE1 tubules ( defined by circularity < 0 . 5 ; n = 2 , NAx = 116 NDd = 134 of siControl carriers , NAx = 71 NDd = 134 of siBin1 carriers ; * P = 0 . 0105 siBin1 - axons compared to siControl - axons , t - , mean ± ) . ( D ) of average ( μm2 ) of BACE1 carriers in axons in dendrites of siBin1 - or siControl - treated neurons ( n = 2 , NAx = 116 NDd = 134 siControl , NAx = 71 NDd = 134 siBin1 ; * * P = 0 . 0019 , Mann - Whitney , mean ± ) .

O O O B-MEASURED_VAR 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 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 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 B-MEASURED_VAR 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 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

(A) BACE1 carriers in axons of siBin1- or siControl-treated neurons expressing BACE1-GFP, analysed by epifluorescence microscopy. Scale bar, 10 µm. White rectangles indicate a BACE1-positive carrier magnified on the right. Scale bar, 1 µm.(B) Quantification of the percentage of siBin1- or siControl-treated neurons displaying extended BACE1 carriers (as shown in (a)); (n=3, NsiControl=67 cells, NsiBin1=68 cells; *P=0.0199, t-test, mean ± SEM).(C) Quantification of circularity of individual BACE1 carriers in axons and in dendrites of siBin1- or siControl-treated neurons. The percentage of BACE1 tubules (defined by circularity < 0.5; n=2, NAx=116 and NDd=134 of siControl carriers, NAx=71 and NDd=134 of siBin1 carriers; *P=0.0105 siBin1-axons compared to siControl-axons, t-test, mean ± SEM).(D) Quantification of average size (μm2) of individual BACE1 carriers in axons and in dendrites of siBin1- or siControl-treated neurons (n=2, NAx=116 and NDd=134 siControl, NAx=71 and NDd=134 siBin1; **P=0.0019, Mann-Whitney test, mean ± SEM).


( E F ) BACE1 ( green ) exit in tubular carriers from Rab5 - positive early endosomes ( magenta ) in axons of siBin1 - ( f ) siControl - ( e ) treated neurons expressing BACE1 - GFP Rab5 - mCherry using time - lapse spinning - disk confocal microscopy ( 4 fps ) ( see EV movie 3 ) . BACE1 Rab5 are shown in axons at 0 sec 4 . 75 sec . Scale bars , 10 µm . rectangles indicate region used in kymographs shown on right . Arrows kymographs ( covering 3 or 4 . 75 sec ) of merged BACE1 Rab5 or BACE1 alone indicate in ( E ) a BACE1 punctum that exits in a tubular carrier from a Rab5 - positive early endosome ( asterisk ) in ( F ) a BACE1 tubule that emanates from a Rab5 - positive early endosome . Scale bars , 1

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

(E and F) BACE1 (green) exit in tubular carriers from Rab5-positive early endosomes (magenta) in axons of siBin1- (f) and siControl- (e) treated neurons expressing BACE1-GFP and Rab5-mCherry using time-lapse spinning-disk confocal microscopy (4 fps) (see EV movie 3). BACE1 and Rab5 are shown in axons at 0 sec and 4.75 sec. Scale bars, 10 µm. White rectangles indicate the region used in kymographs shown on the right. Arrows and kymographs (covering 3 or 4.75 sec) of merged BACE1 and Rab5 or BACE1 alone indicate in (E) a BACE1 punctum that exits in a tubular carrier from a Rab5-positive early endosome (asterisk) and in (F) a BACE1 tubule that emanates from a Rab5-positive early endosome. Scale bars, 1 µm


( A ) APP detected with anti - APP ( Y188 , green ) at early endosomes ( anti - EEA1 , magenta ) in dendrites of siCD2AP - siControl - treated neurons with or without DAPT treatment , analysed by spinning - disk confocal microscopy . Scale bar , 10 µm . squares are magnified on right panels . Scale bar , 1 µm . ( B ) of colocalisation between APP EEA1 - positive dendritic endosomes ( n = 3 , NsiControl DMSO = 15 , NsiControl DAPT = 14 ; NsiCD2AP DMSO = 21 , NsiCD2AP DAPT = 22 ; * * * * P < 0 . 0001 siCD2AP DMSO vs . siControl DMSO , # # # # P < 0 . 0001 siCD2AP DAPT vs . siCD2AP DMSO , t - , mean ± ) .

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

(A) APP detected with anti-APP (Y188, green) at early endosomes (anti-EEA1, magenta) in dendrites of siCD2AP- and siControl-treated neurons with or without DAPT treatment, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares are magnified on the right panels. Scale bar, 1 µm.(B) Quantification of colocalisation between APP and EEA1-positive dendritic endosomes (n=3, NsiControl DMSO=15, NsiControl DAPT=14; NsiCD2AP DMSO=21, NsiCD2AP DAPT=22; ****P<0.0001 siCD2AP DMSO vs. siControl DMSO, ####P<0.0001 siCD2AP DAPT vs. siCD2AP DMSO, t-test, mean ± SEM).


( C ) APP ( green ) distribution within enlarged Rab5QL - GFP endosomes ( magenta ) in dendrites of siCD2AP - or siControl - treated neurons expressing APP - RFP Rab5QL - GFP upon DAPT treatment , analysed by spinning - disk confocal microscopy . Scale bar , 10 µm . squares indicate a Rab5QL - positive endosome magnified on right . Scale bar , 1 µm . ( D ) Qualitative of APP distribution between lumen of Rab5QL - endosomes ( n = 4 , NsiControl = 45 ( 774 endosomes ) , NsiCD2AP = 52 ( 888 endosomes ) ; * P = 0 . 0368 siCD2AP vs . siControl , t - , mean ± ) .

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

(C) APP (green) distribution within enlarged Rab5QL-GFP endosomes (magenta) in dendrites of siCD2AP- or siControl-treated neurons expressing APP-RFP and Rab5QL-GFP upon DAPT treatment, analysed by spinning-disk confocal microscopy. Scale bar, 10 µm. White squares indicate a Rab5QL-positive endosome magnified on the right. Scale bar, 1 µm.(D) Qualitative analysis of APP distribution between the lumen and the membrane of Rab5QL-endosomes (n=4, NsiControl=45 (774 endosomes), NsiCD2AP=52 (888 endosomes); *P=0.0368 siCD2AP vs. siControl, t-test, mean ± SEM).


( E - F ) APP ( green ) distribution within enlarged Rab5QL - GFP endosomes ( magenta ) in bodies of siCD2AP - ( f ) or siControl - ( e ) treated neurons expressing APP - RFP , upon DAPT treatment , analysed by spinning - disk confocal microscopy . APP ( green ) Rab5QL ( magenta ) profiles along endosome ( see inset ) are shown on right . Scale bars , 1 µm . ( G ) of amount of APP at limiting endosomal normalized to total APP per endosome ( n = 3 , NsiControl = 32 endosomes ( 11 ) , NsiCD2AP = 39 endosomes ( 15 ) ; * * * * P < 0 . 0001 , t - , mean ± ) .

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

(E - F) APP (green) distribution within enlarged Rab5QL-GFP endosomes (magenta) in cell bodies of siCD2AP- (f) or siControl- (e) treated neurons expressing APP-RFP, upon DAPT treatment, analysed by spinning-disk confocal microscopy. APP (green) and Rab5QL (magenta) line intensity profiles along the endosome (see inset line) are shown on the right. Scale bars, 1 µm.(G) Quantification of the amount of APP fluorescence at the limiting endosomal membrane normalized to total APP fluorescence per endosome (n=3, NsiControl=32 endosomes (11 cells), NsiCD2AP=39 endosomes (15 cells); ****P<0.0001, t-test, mean ± SEM).


( H ) APP detected with anti - APP ( Y188 , green ) at early endosomes ( anti - EEA1 , magenta ) in dendrites of siCD2AP - siControl - treated neurons upon DAPT treatment , analysed by super - resolution dSTORM imaging . Scale bars , 200 nm .

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

(H) APP detected with anti-APP (Y188, green) at early endosomes (anti-EEA1, magenta) in dendrites of siCD2AP- and siControl-treated neurons upon DAPT treatment, analysed by super-resolution dSTORM imaging. Scale bars, 200 nm.


( I ) Qualitative of super - resolved APP distribution between lumen of EEA1 - positive endosomes of siCD2AP - or siControl - treated neurons upon DAPT treatment ( n = 3 , NsiControl = 35 , NsiCD2AP = 57 , * * P = 0 . 0012 , Mann - Whitney , mean ± ) .

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

(I) Qualitative analysis of super-resolved APP distribution between the lumen and the membrane of EEA1-positive endosomes of siCD2AP- or siControl-treated neurons upon DAPT treatment (n=3, NsiControl=35, NsiCD2AP=57, **P=0.0012, Mann-Whitney test, mean ± SEM).


( J ) of of APP present in EEA1 - positive early endosomes to its centroid over endosome radius . closer is to 1 closer APP is to endosomal ( i . e . , equals radius ) ( n = 3 , NsiControl = 6 , NsiCD2AP = 6 ; * * P = 0 . 0051 , t - , mean ± ) .

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

(J) Quantification of the distance of APP puncta present in EEA1-positive early endosomes to its centroid over the endosome radius. The closer the ratio is to 1 the closer APP is to the endosomal membrane (i.e., distance equals radius) (n=3, NsiControl=6, NsiCD2AP =6; **P=0.0051, t-test, mean ± SEM).


( B ) Direct sequencing of PARN in a control , P1 her parents .

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

(B) Direct sequencing of PARN in a control, P1 and her parents.


( D ) Direct sequencing of PARN in a control , P2 her parents .

(D) Direct sequencing of PARN in a control, P2 and her parents.


( E ) Aberrant splicing products detected in P2 her mother but not in P2 ' s father nor in a healthy control . GAPDH RT - PCR was performed as control .

O O O O O O O O O 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) Aberrant splicing products detected in P2 and her mother but not in P2's father nor in a healthy control. GAPDH RT-PCR was performed as control.


( F ) Detection of exon6 / 7 deletion by PCR with specific primers ( 1F / 2R ) .

(F) Detection of the exon6/7 deletion by PCR with specific primers (1F/2R).


( G ) PARN detection in lysates from P1 P2 a healthy control . Actin was used as loading control .

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(G) PARN detection in cell lysates from P1 and P2 and a healthy control. Actin was used as loading control.


( H ) In vitro deadenylation activity using extracts from control P1 P2 SV40T - transformed fibroblasts . Mixing extracts from control together with P1 or P2 eliminated deadenylation defect whereas pairwise mixing of P1 P2 failed to complement deadenylation defect .

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(H) In vitro deadenylation activity using protein extracts from control and P1 and P2 SV40T-transformed fibroblasts. Mixing protein extracts from control together with P1 or P2 eliminated the deadenylation defect whereas pairwise mixing of P1 and P2 failed to complement the deadenylation defect.


( A ) Mean telomere ( kb ) of whole blood from their parents estimated with TRF method .

(A) Mean telomere length (kb) of whole blood cells from patients and their parents estimated with the TRF method.


( B ) Representative picture of telomeric signals used for Q - FISH on metaphase spreads from SV40T - transfomed fibroblasts Muntjac used to normalize signals . ( C ) mean values of Q - FISH analyses for control . Mean of Muntjac was set to 1 . Error bars indicate s . d . . ( D ) Graphical representation of ( C ) showing average of ratios ( relative to Muntjac ) . Error bars indicate s . e . m . . non - parametric Kruskal - Wallis was applied to compare relative values between Ctl P1 , Ctl P2 .

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(B) Representative picture of telomeric signals used for Q-FISH analysis on metaphase spreads from SV40T-transfomed fibroblasts and Muntjac cells used to normalize the signals. (C) Individual and mean values of Q-FISH analyses for control and patients. Mean fluorescence intensity of Muntjac cells was set to 1. Error bars indicate s.d.. (D) Graphical representation of (C) showing the average of fluorescence ratios (relative to Muntjac). Error bars indicate s.e.m.. The non-parametric Kruskal-Wallis test was applied to compare relative fluorescence values between Ctl and P1, and Ctl and P2.


( E ) Representative pictures of nuclei from two controls ' s fibroblasts showing 53BP1 foci ( green ) telomeres ( red , detected by Telo - FISH ) . Yellow arrows indicate telomere dysfunction - induced foci ( TIF ) . scale bar corresponds to 5µm . ( F ) of ( E ) in fibroblasts from two healthy controls ( passages > 4 ) from 1 ( passages < 4 ) . Control 1 : n = 224 ; Control 2 : n = 263 ; 1 : n = 231 . Averages are shown error bars indicate s . e . m . . Unpaired Student ' s t were applied when indicated .

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(E) Representative pictures of nuclei from two controls and patient's primary fibroblasts showing 53BP1 foci (green) and telomeres (red, detected by Telo-FISH). Yellow arrows indicate telomere dysfunction-induced foci (TIF). The scale bar corresponds to 5µm. (F) Quantification of (E) in primary fibroblasts from two healthy controls (passages > 4) and from patient 1 (passages < 4). Control 1: n = 224; Control 2: n = 263; Patient 1: n = 231. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied when indicated.


( G ) Representative pictures of SA - β - galactosidase in control ( passage 15 ) P1 ' s fibroblasts ( passage 6 ) . Results are expressed as of SA - β - galactosidase - positive ( averages , lower panel ) . Control : n = 436 ; P1 : n = 436 . A to compare two population proportions was applied .

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(G) Representative pictures of SA-β-galactosidase staining in control (passage 15) and P1's primary fibroblasts (passage 6). Results are expressed as the percentage of SA-β-galactosidase-positive cells (averages, lower panel). Control: n = 436; P1: n = 436. A test to compare two population proportions was applied.


( H ) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo - FISH . scale bar corresponds to 1µm . ( I ) of terminal deletions sister telomere losses ( J ) from three independent experiments ( counted chromatids : Ctl1 : n = 2224 ; Ctl2 : n = 5696 ; P1 : n = 4764 ; P2 : n = 7908 ) . Averages are shown χ2 - were applied to compare Ctl1 / 2 with either P1 or P2 .

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(H) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo-FISH. The scale bar corresponds to 1µm. (I) Quantification of terminal deletions and sister telomere losses (J) from three independent experiments (counted chromatids: Ctl1: n=2224; Ctl2: n= 5696; P1: n=4764; P2: n=7908). Averages are shown and χ2-tests were applied to compare Ctl1/2 with either P1 or P2.


( A ) Validation of PARN KO in HT1080 by Western blot . GAPDH is used as loading control .

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(A) Validation of PARN KO in HT1080 cells by Western blot. GAPDH is used as loading control.


( B ) In vitro deadenylation activity using extracts from HT1080 ( WT ) HT1080PARN KO ( PARN KO ) .

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(B) In vitro deadenylation activity assay using protein extracts from HT1080 (WT) and HT1080PARN KO (PARN KO) cells.


( C ) TRAP using successive dilutions ( 500 ng , 250 ng , 125 ng 62 . 5 ng ) of extracts from WT or PARN KO . An internal control ( IC ) for PCR was used .

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(C) TRAP assay using successive dilutions (500 ng, 250 ng, 125 ng and 62.5 ng) of cell extracts from WT or PARN KO cells. An internal control (IC) for PCR was used.


( D ) qRT - PCR of hTR expression in WT PARN KO . Expression were normalized first to ACTB then to WT . Three independent extractions were performed for each . Error bars indicate s . e . m . .

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(D) qRT-PCR analysis of hTR expression in WT and PARN KO cells. Expression levels were normalized first to ACTB and then to WT. Three independent RNA extractions were performed for each cell line. Error bars indicate s.e.m..


( E ) Representative pictures of FISH against hTR in WT PARN KO . Arrows indicate hTR foci . Scale bar 5 μm .

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(E) Representative pictures of FISH against hTR in WT and PARN KO cells. Arrows indicate hTR foci. Scale bar 5 μm.


( F ) PARN detection by Western blot in indicated conditions . Expression was induced by incubating with 10 ng / ml doxycycline for 72 h . Actin is used as loading control . PARN / actin ratios , normalized to doxycycline - treated WT / PARN , are shown below .

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(F) PARN detection by Western blot in the indicated conditions. Expression was induced by incubating cells with 10 ng/ml doxycycline for 72 h. Actin is used as loading control. PARN/actin ratios, normalized to doxycycline-treated WT/PARN cells, are shown below.


( G ) In vitro deadenylation activity using extracts from doxycycline - treated WT / empty , WT / PARN , KO / empty KO / PARN .

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(G) In vitro deadenylation activity assay using protein extracts from doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells.


( H ) qRT - PCR of indicated gene transcripts in doxycycline - treated WT / empty , WT / PARN , KO / empty KO / PARN . Expression were normalized first to ACTB then to WT / empty . Three independent doxycycline inductions were performed for each . Averages are shown error bars indicate s . e . m . . Unpaired Student ' s t were applied .

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(H) qRT-PCR analysis of the indicated gene transcripts in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to WT/empty. Three independent doxycycline inductions were performed for each cell line. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.


( I ) Representative western blot of PARN , TRF2 p53 . Actin was used as loading control . Mean TRF2 / Actin from two independent experiments are indicated below .

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(I) Representative western blot analysis of PARN, TRF2 and p53. Actin was used as loading control. Mean TRF2/Actin levels from two independent experiments are indicated below.


( J ) Same as ( H ) for p53 p21 transcripts . Averages are shown error bars indicate s . e . m . . Unpaired Student ' s t were applied .

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(J) Same as (H) for p53 and p21 transcripts. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.


( K ) qRT - PCR of TERRA from indicated chromosome ends in doxycycline - treated KO / empty KO / PARN . Expression were normalized first to ACTB then to KO / empty . Three independent doxycycline inductions were performed for each . Error bars indicate s . e . m . .

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(K) qRT-PCR analysis of TERRA from the indicated chromosome ends in doxycycline-treated KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to KO/empty. Three independent doxycycline inductions were performed for each cell line. Error bars indicate s.e.m..


( A ) of telomeric aberrations by Telo - FISH in doxycycline - treated WT / empty , WT / PARN , KO / empty KO / PARN . Two independent experiments were performed . Counted chromatids : WT / empty : n = 1824 ; WT / PARN : n = 1744 ; KO / empty : n = 1452 ; KO / PARN : n = 1920 . Averages are shown χ2 - were applied to compare KO / empty with either WT / empty or KO / PARN .

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(A) Analysis of telomeric aberrations by Telo-FISH in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Two independent experiments were performed. Counted chromatids: WT/empty: n=1824; WT/PARN: n= 1744; KO/empty: n=1452; KO/PARN: n=1920. Averages are shown and χ2-tests were applied to compare KO/empty with either WT/empty or KO/PARN.


( B ) TRF of telomere ( kb ) in KO / empty KO / PARN treated with 10 ng / ml Dox for 72h . A control Western blot with PARN Actin is shown below for corresponding samples .

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(B) TRF analysis of telomere length (kb) in KO/empty and KO/PARN cells treated with 10 ng/ml Dox for 72h. A control Western blot with PARN and Actin is shown below for the corresponding samples.


( A ) HT1080 ( WT ) HT1080PARN KO ( KO ) stably transfected with GFP plasmid ( empty ) were transfected with siRNAs against p53 for 72h before extraction qRT - PCR . siRNAs against luciferase ( siLuci ) were used as control . p53 expression were normalized first to ACTB then to WT / empty / siLuci . Experiment was performed in triplicate . Error bars indicate s . e . m .

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(A) HT1080 (WT) and HT1080PARN KO (KO) cells stably transfected with GFP plasmid (empty) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT-PCR. siRNAs against luciferase (siLuci) were used as control. p53 expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.


( B ) were treated as in ( A ) transcript of indicated genes were measured by qRT - PCR . cDNA expression were normalized first to ACTB then to WT / empty / siLuci . Experiment was performed in triplicate . Error bars indicate s . e . m .

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(B) Cells were treated as in (A) and transcript levels of the indicated genes were measured by qRT-PCR. cDNA expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.


( C ) qRT - PCR analyses of indicated genes were performed on extracted from P1 ' s or SV40T - transformed fibroblasts . cDNA expression were normalized to fibroblasts .

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(C) qRT-PCR analyses of the indicated genes were performed on RNA extracted from P1's primary or SV40T-transformed fibroblasts. cDNA expression levels were normalized to primary fibroblasts.


( C ) Northern blot of pre - rRNAs from control B - LCLs . ( D ) Log2 values of 18S - E / 21S 18S - EFL / 21S for P1 P2 were normalized to values of control presented in a graphical format , as Log averages + standard deviation for two independent experiments .

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(C) Northern blot analysis of pre-rRNAs from control and patient B-LCLs. (D) Log2 values of 18S-E/21S and 18S-EFL/21S for P1 and P2 were normalized to the values of the control and presented in a graphical format, as Log averages + standard deviation for two independent experiments.


( E ) Northern blot of pre - rRNAs from HT1080PARN WT HT1080PARN KO , transduced with an empty vector . ( F ) Ectopic expression of PARN was induced in HT1080PARN KO compared to transduced with an empty vector . Western blot relative to actin assessed PARN . ( G ) Quantitative analyses of 18S - E / 21S 18S - E FL / 21S ratios were as described in ( D ) for HT1080PARN KO relative to HT1080PARN WT ( left panel ; see ( E ) ) , for HT1080PARN KO rescued by ectopic expression of PARN relative to HT1080PARN KO transduced with an empty expression vector ( right panel ; see ( F ) ) , with Log averages + standard deviation for four independent experiments .

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(E) Northern blot analysis of pre-rRNAs from HT1080PARN WT and HT1080PARN KO cells, transduced with an empty vector. (F) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector. Western blot relative to actin assessed PARN levels. (G) Quantitative analyses of 18S-E/21S and 18S-E FL/21S ratios were as described in (D) for HT1080PARN KO cells relative to HT1080PARN WT (left panel; see (E)), and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector (right panel; see (F)), with Log averages + standard deviation for four independent experiments.


( A ) Western blot of Parn expression in control Parn + / - MEFs .

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(A) Western blot analysis of Parn expression in control and Parn+/- MEFs.


( B ) Northern blot of total RNAs from WT Parn + / - MEFs .

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(B) Northern blot analysis of total RNAs from WT and Parn+/- MEFs.


( D ) Frequencies of expected observed newborns of indicated genotype obtained from Parn + / - intercrosses . n indicates number of analyzed . Statistical significance ( P = 1 . 5417E - 08 ) of differences between observed expected genotype distributions was assessed by χ2 - . ( E ) Frequencies of expected observed E11 . 5 of indicated genotype obtained from Parn + / - intercrosses . n indicates number of analysed . Statistical significance ( P = 0 . 02535 ) of differences between observed expected genotype distributions was assessed by χ2 - .

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(D) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analyzed. Statistical significance (P = 1.5417E-08) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (E) Frequencies of expected and observed E11.5 embryos of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analysed. Statistical significance (P = 0.02535) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


( F ) , ( G ) p53 knock - out does not rescue Parn - / - . No Parn - / - p53 - / - mouse was born from various combinations of Parn + / - either p53 + / - or p53 - / - crosses ( 49 56 pups , respectively ) . Statistical significance ( P = 0 . 000886 ) of differences between observed expected genotype distributions was assessed by χ2 - .

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(F), (G) p53 knock-out does not rescue Parn-/-. No Parn-/- p53-/- mouse was born from various combinations of Parn+/- and either p53+/- or p53-/- crosses (49 and 56 pups, respectively). Statistical significance (P = 0.000886) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


( C ) Streptavidin Western blotting of total biotinylated in APEX2 : RAB or APEX2 only Flp - In / T - REx HeLa .

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(C) Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp-In/T-REx HeLa cells.


( D ) APEX2 : RAB biotinylated ( streptavidin ) are partially colocalized with EEA1 in HeLa , n = 2 independent experiments . Scale bars : 10µm or 5µm in enlarged views .

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(D) APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n=2 independent experiments. Scale bars: 10µm or 5µm in enlarged views.


( C ) ProHits - viz generated dotblots of RAB effectors , GEFs , GAPs sorting complexes . Green , blue beige shadings along with RAB names refer to previously identified interactions between RABs depicted .

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(C) ProHits-viz generated dotblots of RAB effectors, GEFs, GAPs and sorting complexes. Green, blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins.


Strumpellin VPS35 are in close proximity to RAB5 RAB21 . ( A ) Anti - biotin immunoprecipitation immunoblot of endogenous Strumpellin VPS35 . Lysates correspond to 1 % of input , n = 3 independent experiments . RAB21 can be seen interacting with various WASH retromer complexes subunits .

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Strumpellin and VPS35 are in close proximity to RAB5 and RAB21. (A) Anti-biotin immunoprecipitation and immunoblot of endogenous Strumpellin and VPS35. Lysates correspond to 1% of input, n=3 independent experiments. RAB21 can be seen interacting with various WASH and retromer complexes subunits.


GFP trap IP of WT , Q78L T33N RAB21 variants in HeLa followed by GFP immunoblot endogenous ( B ) VPS26 immunoblot Lysates correspond to 5 % of input , n ≥ 3 independent experiments .

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GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and endogenous (B) VPS26 immunoblot Lysates correspond to 5% of input, n≥ 3 independent experiments.


GFP trap IP of WT , Q78L T33N RAB21 variants in HeLa followed by GFP immunoblot ( C ) FAM21 , Strumpellin VPS35 immunoblots . Lysates correspond to 5 % of input , n ≥ 3 independent experiments .

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GFP trap IP of WT, Q78L and T33N RAB21 variants in HeLa cells followed by GFP immunoblot and (C) FAM21, Strumpellin and VPS35 immunoblots. Lysates correspond to 5% of input, n≥ 3 independent experiments.


RAB21 actively pull - downs WASH retromer subunits ( D ) Bacterially - purified GTP - loaded GST : RAB21 pull - down of HeLa lysates followed by Strumpellin , FAM21 , CAPZα VPS35 immunoblots . Ponceau reveals purity of GST GST : RAB21 used for pull - downs . n = 3 independent experiments .

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RAB21 actively pull-downs WASH and retromer complex subunits (D) Bacterially-purified and GTP-loaded GST:RAB21 pull-down of HeLa cell lysates followed by Strumpellin, FAM21, CAPZα and VPS35 immunoblots. Ponceau staining reveals the purity of the GST and GST:RAB21 used for the pull-downs. n=3 independent experiments.


( E ) RAB21 colocalizes with WASH retromer complexes . Transiently expressing GFP : RAB21 HeLa were fixed stained for endogenous EEA1 , FAM21 , WASH VPS26 . Boxed region is magnified single channels are depicted . Scale bars : 10µm or 5µm in enlarged view .

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(E) RAB21 colocalizes with the WASH and retromer complexes. Transiently expressing GFP:RAB21 HeLa cells were fixed and stained for endogenous EEA1, FAM21, WASH and VPS26. Boxed region is magnified and single channels are depicted. Scale bars: 10µm or 5µm in enlarged view.


( F ) Per Pearson Correlation between RAB21 various markers . Error bars are , n = 2 independent experiments .

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(F) Per cell Pearson Correlation between RAB21 and the various markers. Error bars are SEM, n=2 independent experiments.


( A ) Low RAB21 expression in two independent RAB21 KO populations . Immunoblot of endogenous RAB21 , RAB5 , RAB7 GAPDH .

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(A) Low RAB21 expression levels in two independent RAB21 KO cell populations. Immunoblot analysis of endogenous RAB21, RAB5, RAB7 and GAPDH.


( B ) Endo - lysosomal compartments are unaffected by loss of RAB21 . Immunofluorescences of APPL1 , TfR , RAB7 LAMP1 in parental HeLa in two RAB21 knockout populations . Scale bars : 10µm , n = 3 independent experiments .

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(B) Endo-lysosomal compartments are unaffected by the loss of RAB21. Immunofluorescences of APPL1, TfR, RAB7 and LAMP1 in parental HeLa cells and in the two RAB21 knockout cell populations. Scale bars :10µm, n=3 independent experiments.


stability of retromer cargo sorting is affected in RAB21 KO . Immunoblot of VPS35 , VPS26 , VPS29 , GAPDH RAB21 in parental two RAB21 KO populations . of VPS35 , VPS26 VPS29 integrated densities to GAPDH for four independent experiments ; . * represents p < 0 . 05 Statistical used : One sample t - error bars are . Number of presented on each graph represents total number of analyzed for repeats .

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The stability of the retromer cargo sorting complex is affected in RAB21 KO cells. Immunoblot analysis of VPS35, VPS26, VPS29, GAPDH and RAB21 in parental and the two RAB21 KO cell populations. Ratio of VPS35, VPS26 and VPS29 integrated densities to GAPDH for four independent experiments; SEM. * represents p <0.05 Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( B ) WASH are not affected by RAB21 deletion . Immunoblot of Strumpellin , WASH , FAM21 , CAPZα , GAPDH RAB21 . of WASH , FAM21 , Strumpellin CAPZα integrated densities to GAPDH for four independent experiments ; . Data information Statistical used : One sample t - error bars are . Number of presented on each graph represents total number of analyzed for repeats .

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(B) WASH complex proteins are not affected by RAB21 deletion. Immunoblot of Strumpellin, WASH, FAM21, CAPZα, GAPDH and RAB21. Ratio of WASH, FAM21, Strumpellin and CAPZα integrated densities to GAPDH for four independent experiments; SEM. Data information Statistical tests used: One sample t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


RAB21 knockout decreases VPS35 localization at endosomes . ( C ) Immunofluorescence of endogenous VPS35 EEA1 in RAB21 - depleted . Boxed region is magnified single channel are depicted . ( D ) Per Pearson Correlation between VPS35 EEA1 ; , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . Statistical used : ( D ) - Unpaired t - error bars are . Number of presented on each graph represents total number of analyzed for repeats .

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RAB21 knockout decreases VPS35 localization at endosomes. (C) Immunofluorescence of endogenous VPS35 and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (D) Per cell Pearson Correlation between VPS35 and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. Statistical tests used: (D) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


RAB21 knockout decreases VPS35 localization at endosomes . ( E ) Average number of VPS35 per ; , n = 3 independent experiments . Data information Statistical used : Mann - Whitney . error bars are . Number of presented on each graph represents total number of analyzed for repeats .

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RAB21 knockout decreases VPS35 localization at endosomes. (E) Average number of VPS35 puncta per cell; SEM, n=3 independent experiments. Data information Statistical tests used: Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.


( F - H ) RAB21 loss impairs endosomal recruitment of WASH . ( F ) Immunofluorescence of endogenous WASH EEA1 in RAB21 - depleted . Boxed region is magnified single channel are depicted . ( G ) Per Pearson Correlation between VPS35 EEA1 ( H ) FAM21 EEA1 ; , n = 2 independent experiments . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views . error bars are . Number of presented on each graph represents total number of analyzed for repeats .

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(F-H) RAB21 loss impairs endosomal recruitment of the WASH complex. (F) Immunofluorescence of endogenous WASH and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (G) Per cell Pearson Correlation between VPS35 and EEA1 and (H) FAM21 and EEA1; SEM, n=2 independent experiments. Data information: scale bars represent 10µm or 2.5µm in enlarged views. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.