( 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 ) .

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 O O O 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 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-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 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 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-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

(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 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 ) .

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 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 O O O O O B-MEASURED_VAR 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

(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 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 ) .

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

O 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 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 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 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

(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 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 ) .

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 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 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 O B-MEASURED_VAR 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

(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 ) 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 )

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

(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 ) 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 ) .

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

(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 . 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 ) .

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

(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 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

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 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 B-MEASURED_VAR 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 B-MEASURED_VAR O B-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 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 - 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 ) .

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 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 B-MEASURED_VAR 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 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 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 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 ) .

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 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 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 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 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 ) .

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 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 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 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

(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 - and 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 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 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 ) .

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 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 ) 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 ) .

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 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

(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 and her parents .

O O O O O O B-MEASURED_VAR O 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 and her parents .

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


( 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 .

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

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


( G ) PARN detection in cell lysates from P1 and P2 and 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 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 .

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

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(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 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 cells 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 assay using protein extracts from HT1080 ( WT ) and HT1080PARN KO ( PARN KO ) cells .

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


( 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 .

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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 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 . .

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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 and PARN KO cells . 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 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 .

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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 assay using protein extracts from doxycycline - treated WT / empty , WT / PARN , KO / empty and KO / PARN cells .

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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 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 .

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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 analysis of PARN , TRF2 and p53 . Actin was used as loading control . Mean TRF2 / Actin levels 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 and p21 transcripts . Averages are shown and error bars indicate s . e . m . . Unpaired Student ' s t tests 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 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 . .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 the indicated genes were performed on RNA extracted from P1 ' s primary or SV40T - transformed fibroblasts . cDNA expression levels were normalized to primary 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 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 .

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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 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 .

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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 analysis of Parn expression in control and Parn + / - MEFs .

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


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

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


( 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 .

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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 + / - 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 .

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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 proteins in APEX2 : RAB or APEX2 only Flp - In / T - REx HeLa cells .

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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 proteins ( streptavidin ) are partially colocalized with EEA1 in HeLa cells , 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 and sorting complexes . Green , blue and beige shadings along with RAB names refer to previously identified interactions between RABs and the depicted proteins .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cell Pearson Correlation between RAB21 and the various markers . Error bars are SEM , 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 levels in two independent RAB21 KO cell populations . Immunoblot analysis of endogenous RAB21 , RAB5 , RAB7 and 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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


( I - J ) RAB21 knockout reduces endosomal F - actin levels . ( I ) Immunofluorescence of endogenous F - actin using Alexa - 488 conjugated phalloidin and EEA1 in RAB21 - depleted cells . Boxed region is magnified and single channel images are depicted . ( J ) Per cell Pearson Correlation between internal F - actin and EEA1 ; SEM , n = 3 independent experiments . Data information scale bars represent 10µm or 2 . 5µm in enlarged views . 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 .

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(I-J) RAB21 knockout reduces endosomal F-actin levels. (I) Immunofluorescence of endogenous F-actin using Alexa-488 conjugated phalloidin and EEA1 in RAB21-depleted cells. Boxed region is magnified and single channel images are depicted. (J) Per cell Pearson Correlation between internal F-actin and EEA1; SEM, n=3 independent experiments. Data information scale bars represent 10µm or 2.5µm in enlarged views. 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.


( A - C ) RAB21 is not required for CI - MPR or GLUT1 trafficking . ( A ) Immunofluorescence of endogenous CI - MPR and TGN46 or GLUT1 with LAMP1 in RAB21 - knockout cells . Boxed region is magnified . ( B ) Per cell Pearson Correlation between CI - MPR and TGN46 or ( C ) GLUT1 and LAMP1 ; SEM , n = 3 independent experiments . Data information : In ( A ) , scale bars represent 20µm or 5µm in enlarged views for CI - MPR , while they represent 10µm in enlarged views for GLUT1 . Statistical tests used : ( B ) - Unpaired t - tests ( C Mann - Whitney tests . All error bars are SEM . Number of cells presented on each graph represents the total number of cells analyzed for all

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(A-C) RAB21 is not required for CI-MPR or GLUT1 trafficking. (A) Immunofluorescence of endogenous CI-MPR and TGN46 or GLUT1 with LAMP1 in RAB21-knockout cells. Boxed region is magnified. (B) Per cell Pearson Correlation between CI-MPR and TGN46 or (C) GLUT1 and LAMP1; SEM, n=3 independent experiments. Data information: In (A), scale bars represent 20µm or 5µm in enlarged views for CI-MPR, while they represent 10µm in enlarged views for GLUT1. Statistical tests used: (B) - Unpaired t-tests (C 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


MCT1 trafficking requires RAB21 . ( D ) Immunofluorescence of endogenous MCT1 in wild type or RAB21 - depleted cells . ( E ) Per cell integrated MCT1 intensity ( in Relative Fluorescence Unit ) ; SEM , n = 2 independent experiments . Data information : scale bars represent 20µm or 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

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MCT1 trafficking requires RAB21. (D) Immunofluorescence of endogenous MCT1 in wild type or RAB21-depleted cells. (E) Per cell integrated MCT1 intensity (in Relative Fluorescence Unit); SEM, n=2 independent experiments. Data information: scale bars represent 20µm or 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


( F ) RAB21 interacts with SLC3A2 . FLAG immunoprecipitation of FLAG : RAB21 and immunoblot of co - expressed SLC3A2 : HA . Lysates correspond to 5 % input , n = 3 independent experiments .

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(F) RAB21 interacts with SLC3A2. FLAG immunoprecipitation of FLAG:RAB21 and immunoblot of co-expressed SLC3A2:HA. Lysates correspond to 5% input, n=3 independent experiments.


( G - H ) RAB21 is required for appropriate SLC3A2 , Basigin and CD44 trafficking . ( G ) Antibody - uptake assays in parental or knockout RAB21 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Note that non - internalized antibodies were removed by an acid wash , which was not fully efficient for CD147 and CD44 , explaining plasma membrane labeling . ( H ) Percentage of cells with tubules in parental or RAB21 knockout cells ; SEM , n = 3 independent experiments . Data information scale bars represent 20µm or 5µm in enlarged views . 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

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(G-H) RAB21 is required for appropriate SLC3A2, Basigin and CD44 trafficking. (G) Antibody-uptake assays in parental or knockout RAB21 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Note that non-internalized antibodies were removed by an acid wash, which was not fully efficient for CD147 and CD44, explaining plasma membrane labeling. (H) Percentage of cells with tubules in parental or RAB21 knockout cells; SEM, n=3 independent experiments. Data information scale bars represent 20µm or 5µm in enlarged views. 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


( A ) Validation of WASH and retromer knockouts and SLC3A2 protein levels . Immunoblots of parental or FAM21 , VARP and VPS29 knockout cell populations .

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(A) Validation of WASH and retromer knockouts and SLC3A2 protein levels. Immunoblots of parental or FAM21, VARP and VPS29 knockout cell populations.


( B ) Antibody - uptake assays in parental or knockout FAM21 , VARP and VPS29 cells . Cells were stained with an Alexa488 conjugated anti - mouse antibody . Arrowheads point to endosomal tubules . Data information : ( B ) scale bars represent

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(B) Antibody-uptake assays in parental or knockout FAM21, VARP and VPS29 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Data information: (B) scale bars represent 20µm


( C ) Percentage of cells with tubules in parental or in various knockout cells ; SEM , n = 3 independent experiments . Statistical tests used : ( C ) - 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 .

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(C) Percentage of cells with tubules in parental or in various knockout cells; SEM, n=3 independent experiments. Statistical tests used: (C) - 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.


( D ) Ratio of SLC3A2 integrated densities to GAPDH ; SEM , n = 3 independent experiments . Statistical tests used : ( D ) 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 .

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(D) Ratio of SLC3A2 integrated densities to GAPDH; SEM, n=3 independent experiments. Statistical tests used: (D) 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.


Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( E ) EEA1 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information scale bars represent 10µm or 2 . 5µm in enlarged views .

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Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (E) EEA1 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information scale bars represent 10µm or 2.5µm in enlarged views.


Retromer is required for endosomal localization of RAB21 . Immunofluorescence of transiently expressed GFP : RAB21 with endogenous ( F ) VPS26 in parental or VARP - and VPS29 - deleted cells . Boxed region is magnified underneath . Data information : scale bars represent 10µm or 2 . 5µm in enlarged views .

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Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (F) VPS26 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information: scale bars represent 10µm or 2.5µm in enlarged views.


( G ) Per cell Pearson Correlation between GFP : RAB21 and EEA1 or VPS26 ; SEM , n = 3 independent experiments . Statistical tests used : ( G ) - 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 .

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(G) Per cell Pearson Correlation between GFP:RAB21 and EEA1 or VPS26; SEM, n=3 independent experiments. Statistical tests used: (G) - 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.


A , C ) After 24 , 48 , and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner . Untreated cells were measured as controls ( n = 12 , mean ± S . D . ) .

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A,C) After 24, 48, and 72 h incubation bortezomib inhibited cell growth in a concentration dependent manner. Untreated cells were measured as controls (n = 12, mean ± S.D.).


B , D ) Dynamic proliferation curves for Cal - 78 and SW - 1353 cells in the presence of 0 ( red ) , 2 . 5 nM ( green ) , 5 nM ( blue ) , and 10 nM ( purple ) bortezomib . Data shown are representatives from three independent experiments ( n = 3 , measured in biological quadruplicates ) .

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B,D) Dynamic proliferation curves for Cal-78 and SW-1353 cells in the presence of 0 (red), 2.5 nM (green), 5 nM (blue), and 10 nM (purple) bortezomib. Data shown are representatives from three independent experiments (n = 3, measured in biological quadruplicates).


A , C ) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3 / 7 activity than untreated cells . Untreated control cells served as reference value ( ratio = 1 ) .

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A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1).


B , D ) Cleavage of caspase - 3 was detected after 24 h of bortezomib treatment by flow cytometry . The y - axis denotes cell counts and the x - axis represents fluorescence intensity of the APC antibody . The black filled histogram represents untreated control cells , the striated histogram represents 5 nM , and the checkered histogram shows 10 nM bortezomib treated cells .

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B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.


A , B ) Relative gene expression analysis of the pro - and anti - apoptotic markers Bax , Bak , Bcl - 2 , and Bcl - xl in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h ( n = 10 ) .

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A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h (n = 10).


C ) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27 , HSP60 , and HSP70 as well as HTRA , Livin , p27 , and p53 in both cell lines .

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C) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27, HSP60, and HSP70 as well as HTRA, Livin, p27, and p53 in both cell lines.


D ) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment .

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D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.


A , B ) Relative gene expression analysis of IGF1R , Fas , and the death receptors TRAILR - 1 and TRAILR - 2 after bortezomib treatment with the respective IC50 concentrations for 48 h . Untreated control cells served as reference value ( ratio = 1 ) .

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A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC50 concentrations for 48 h. Untreated control cells served as reference value (ratio = 1).


C ) The human apoptosis antibody protein array supported the important role of the IGF binding proteins ( IGFBP - 1 to IGFBP - 6 ) , the TNF receptor family , and the TRAIL receptors in both cell lines .

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C) The human apoptosis antibody protein array supported the important role of the IGF binding proteins (IGFBP-1 to IGFBP-6), the TNF receptor family, and the TRAIL receptors in both cell lines.


D ) Western blot analysis showed the downregulation of Fas and TNF - R1 .

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D) Western blot analysis showed the downregulation of Fas and TNF-R1.


A ) Relative gene expression and B ) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5 / 12 and Beclin in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h .

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A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.


B ) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5 / 12 and Beclin in Cal - 78 and SW - 1353 cells treated with the respective IC50 values of bortezomib for 24 h .

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B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h.


C ) Western blot analysis for the expression of LC3BI - II . The lysosomal protease inhibitors E64d and pepstatin A ( Inh ) blocked the autophagic flux and inhibited the degradation of LC3B - II .

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C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II.


D ) Quantification of the relative LC3B protein expression .

D) Quantification of the relative LC3B protein expression.


A ) Effect of bortezomib treatment on LC3B immunostaining of Cal - 78 and SW - 1353 cells . Double immunolabeling with DAPI ( blue ) and anti - LC3B ( green ) antibodies was performed as described in the methods section . The lysosomal protease inhibitors E64d and pepstatin A ( Inh ) blocked the autophagic flux and inhibited the degradation of LC3B - II ( bar : 20 μm ) .

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A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm).


B ) Cal - 78 and SW - 1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A ( Inh ; striated bars ) and cell viability was analysed by the MTS assay . Untreated cells were measured as controls ( light grey ; n = 8 , mean ± S . D . ) .

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B) Cal-78 and SW-1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A (Inh; striated bars) and cell viability was analysed by the MTS assay. Untreated cells were measured as controls (light grey; n = 8, mean ± S.D.).


Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( A ) Age - matched control Atg7 littermates ( Atg7flox / + ; nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) or Atg7 KO ( Atg7flox / flox ; nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) are shown .

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (A) Age-matched control Atg7 littermates (Atg7flox/+;nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4) or Atg7 KO (Atg7flox/flox;nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3) are shown.


Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( B ) Control Atg5 littermates ( Atg5flox / + ; nestin - Cre ; denoted + ) at 16 ( open circles ; n = 4 ) and 26 wk ( open diamonds ; n = 3 ) or Atg5 KO ( Atg5flox / flox ; nestin - Cre ; denoted − ) at 16 ( closed circles ; n = 4 ) and 26 wk ( closed diamonds ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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

Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (B) Control Atg5 littermates (Atg5flox/+;nestin-Cre; denoted +) at 16 (open circles; n = 4) and 26 wk (open diamonds; n = 3) or Atg5 KO (Atg5flox/flox;nestin-Cre; denoted −) at 16 (closed circles; n = 4) and 26 wk (closed diamonds; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Suppressed accumulation of Ub in Atg7 - / p62 - deficient brains . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured total ( A ) and isopeptide - linked ( B ) Ub from the soluble and insoluble fractions of autophagy - deficient brains . Age - matched control Atg7 littermates ( Atg7flox / + : nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) , p62 KO ( Atg7flox / flox : p62 − / − ; denoted − ) at 6 wk ( closed circles ; n = 3 ) , Atg7 KO ( Atg7flox / flox : nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) , or Atg7 - / p62 - DKO ( Atg7flox / flox : nestin - Cre : p62 − / − ) at 6 wk ( closed triangles ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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

Suppressed accumulation of Ub in Atg7-/p62-deficient brains. (A and B) Quantitative mass spectrometry analysis of P2UBA captured total (A) and isopeptide-linked (B) Ub from the soluble and insoluble fractions of autophagy-deficient brains. Age-matched control Atg7 littermates (Atg7flox/+:nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4), p62 KO (Atg7flox/flox:p62−/−; denoted −) at 6 wk (closed circles; n = 3), Atg7 KO (Atg7flox/flox:nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3), or Atg7-/p62-DKO (Atg7flox/flox:nestin-Cre:p62−/−) at 6 wk (closed triangles; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Suppressed accumulation of Ub in Atg7 - / Nrf2 - deficient livers . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured p62 ( A ) , total ( A ) , and isopeptide - linked ( B ) Ub from the soluble and insoluble fraction of autophagy - deficient livers ( 28 d after pIpC injection ) . Control littermates ( F / F or F / F : Nrf2 + / − ; denoted + ; open circles ; n = 4 ) , Nrf2 KO ( F / F : Nrf2 − / − ; denoted − ; closed circles ; n = 4 ) , Atg7 KO ( F / F : Mx1 or F / F : Mx1 : Nrf2 + / − ; denoted − ; closed diamonds ; n = 6 ) , or Atg7 - / Nrf2 - DKO ( F / F : Mx1 : Nrf2 − / − ; closed triangles ; n = 6 ) are shown . Each symbol represents one animal . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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

Suppressed accumulation of Ub in Atg7-/Nrf2-deficient livers. (A and B) Quantitative mass spectrometry analysis of P2UBA captured p62 (A), total (A), and isopeptide-linked (B) Ub from the soluble and insoluble fraction of autophagy-deficient livers (28 d after pIpC injection). Control littermates (F/F or F/F:Nrf2+/−; denoted +; open circles; n = 4), Nrf2 KO (F/F:Nrf2−/−; denoted −; closed circles; n = 4), Atg7 KO (F/F:Mx1 or F/F:Mx1:Nrf2+/−; denoted −; closed diamonds; n = 6), or Atg7-/Nrf2-DKO (F/F:Mx1:Nrf2−/−; closed triangles; n = 6) are shown. Each symbol represents one animal. **, P ≤ 0.01; ***, P ≤ 0.005.


( B ) Elevated Ub mRNA levels from Atg5 - deficient brains . Total RNA was extracted from control Atg5 littermates ( Atg5flox / + ; nestin - Cre ) at 16 wk or Atg5 KO ( Atg5flox / flox ; nestin - Cre ) at 16 wk ( n = 4 for each genotype ; * * , P ≤ 0 . 01 ) and the amount of transcript from Ubc , Ubb , Uba52 , and Uba80 genes was quantified using real - time RT - PCR and normalized to actin . Error bars indicate SEM . au , arbitrary unit .

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(B) Elevated Ub mRNA levels from Atg5-deficient brains. Total RNA was extracted from control Atg5 littermates (Atg5flox/+;nestin-Cre) at 16 wk or Atg5 KO (Atg5flox/flox;nestin-Cre) at 16 wk (n = 4 for each genotype; **, P ≤ 0.01) and the amount of transcript from Ubc, Ubb, Uba52, and Uba80 genes was quantified using real-time RT-PCR and normalized to actin. Error bars indicate SEM. au, arbitrary unit.


( B ) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line , m5 - 7 , as measured by the decreased levels of phosphatidylethanolamine - modified LC3 ( LC3 - II ) .

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(B) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line, m5-7, as measured by the decreased levels of phosphatidylethanolamine-modified LC3 (LC3-II).


( C ) Western blot ( WB ) analysis of the accumulation of htt ( Q ) - GFP after autophagy shutoff . At steady - state ( t = 0 h ) , the levels of both htt ( Q25 ) - GFP and htt ( Q47 ) - GFP were virtually undetectable . After autophagy shutoff , there was a robust increase in the level of htt ( Q47 ) - GFP , as detected by GFP Western blot , whereas there was very little or no detectable increase in htt ( Q25 ) - GFP , GAPDH , LDH , or chFP .

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(C) Western blot (WB) analysis of the accumulation of htt(Q)-GFP after autophagy shutoff. At steady-state (t = 0 h), the levels of both htt(Q25)-GFP and htt(Q47)-GFP were virtually undetectable. After autophagy shutoff, there was a robust increase in the level of htt(Q47)-GFP, as detected by GFP Western blot, whereas there was very little or no detectable increase in htt(Q25)-GFP, GAPDH, LDH, or chFP.


( D ) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells . The ASI is the ratio of the relative increase in green and red fluorescence , as assessed by two - color flow cytometry , in response to dox . In control experiments , the ASI of two soluble , nonaggregating proteins , htt ( Q25 ) - GFP and chFP , was 1 . In contrast , the ASI of the aggregation - prone protein , htt ( Q47 ) - GFP , compared with chFP after autophagy shutoff ( + dox ; 100 h ) was 2 . Data represent three independent experiments performed on different days . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

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(D) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells. The ASI is the ratio of the relative increase in green and red fluorescence, as assessed by two-color flow cytometry, in response to dox. In control experiments, the ASI of two soluble, nonaggregating proteins, htt(Q25)-GFP and chFP, was 1. In contrast, the ASI of the aggregation-prone protein, htt(Q47)-GFP, compared with chFP after autophagy shutoff (+dox; 100 h) was 2. Data represent three independent experiments performed on different days. **, P ≤ 0.01; ***, P ≤ 0.005.


( E ) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff ( + dox ) . The degree of cell death correlated directly with increased htt ( Q47 ) - GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt ( Q47 ) - GFP compared with htt ( Q25 ) - GFP ( * * * , P ≤ 0 . 005 ) . Data represent three independent experiments performed on different days .

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(E) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff (+dox). The degree of cell death correlated directly with increased htt(Q47)-GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt(Q47)-GFP compared with htt(Q25)-GFP (***, P ≤ 0.005). Data represent three independent experiments performed on different days.


( F ) AQUA using heavy isotope - labeled GFP standard in addition to the routine Ub standards after P2UBA pull - down of soluble and insoluble lysates from htt ( Q ) - GFP - IRES - chFP bicistronic expressing cells before ( − dox ) and after autophagy shutoff ( + dox ) . The experimental GFP peptide was only detected in two of the four experiments , and the data shown are representative of these . In the other two experiments , the experimental GFP peptide was below the threshold of detection .

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

(F) AQUA using heavy isotope-labeled GFP standard in addition to the routine Ub standards after P2UBA pull-down of soluble and insoluble lysates from htt(Q)-GFP-IRES-chFP bicistronic expressing cells before (−dox) and after autophagy shutoff (+dox). The experimental GFP peptide was only detected in two of the four experiments, and the data shown are representative of these. In the other two experiments, the experimental GFP peptide was below the threshold of detection.


( A - C ) The relative levels of ( A ) canalicular membrane ( BA ) transporter Bsep , Mrp2 , Mdr1b , Mdr2 , Abcg5 and Abcg8 mRNA transcripts , ( B ) basolateral membrane BA transporter Mrp3 , Mrp4 , Ostβ , Ntcp and Oatp1a1 mRNA transcripts , and ( C ) BA synthetic enzyme Cyp7a1 , Cyp7b1 , Cyp8b1 , Cyp27a1 , and Cyp2c70 mRNA transcripts . Wild type , wild type mice , n = 6 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 7 . Data are shown as means ± SD . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test .

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(A-C) The relative levels of (A) canalicular membrane (BA) transporter Bsep, Mrp2, Mdr1b, Mdr2, Abcg5 and Abcg8 mRNA transcripts, (B) basolateral membrane BA transporter Mrp3, Mrp4, Ostβ, Ntcp and Oatp1a1 mRNA transcripts, and (C) BA synthetic enzyme Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and Cyp2c70 mRNA transcripts. Wild type, wild type mice, n=6; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=7. Data are shown as means ± SD. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.


( D ) Western blot analysis of the relative levels of Bsep , Mrp2 , Mdr2 , Mrp3 , Mrp4 , Ostα / β , Ntcp , Cyp7a1 and Cyp8b1 protein expression in mouse livers . Contrary to the mRNA alterations , the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers , suggesting that the decreased these proteins were might be mediated by post - translational regulation in the livers of Sema7aR145W mutant mice . Wild type , wild type mice , n = 4 ; Heterozygote , Sema7aR145W heterozygous mice , n = 5 ; Homozygote , Sema7aR145W homozygous mice , n = 5 . * P < 0 . 05 versus the WT mice , # P < 0 . 05 versus the heterozygous mutant mice . The data were analyzed by the independent - samples Student ' s t - test and the Mann - Whitney U test .

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

(D) Western blot analysis of the relative levels of Bsep, Mrp2, Mdr2, Mrp3, Mrp4, Ostα/β, Ntcp, Cyp7a1 and Cyp8b1 protein expression in mouse livers. Contrary to the mRNA alterations, the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers, suggesting that the decreased these proteins were might be mediated by post-translational regulation in the livers of Sema7aR145W mutant mice. Wild type, wild type mice, n=4; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=5. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.


( E , F ) Multiplex IF analyses of Bsep and Mrp2 expression in the livers ( E ) and primary hepatocytes in sandwich cultures ( F ) from WT and homozygous mice . Scale bar , 50 µm .

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(E, F) Multiplex IF analyses of Bsep and Mrp2 expression in the livers (E) and primary hepatocytes in sandwich cultures (F) from WT and homozygous mice. Scale bar, 50 µm.


( G ) Multiplex IF analyses revealed that transfection with the SEMA7A _ WT or SEMA7A _ R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose - dependent manner ( 0 , 0 . 2 , 1 . 0 μg per well of 6 - well plate ; 1 . 5 ml culture medium per well ) . Scale bar , 50 µm . Together , the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis ( Appendix Table S16 ) . Subsequently , cholestasis triggered an adaptive response in the liver by down - regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up - regulating the expression of BA efflux transporters Mrp3 , Mrp4 and Ostα / β .

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

(G) Multiplex IF analyses revealed that transfection with the SEMA7A_WT or SEMA7A_R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose-dependent manner (0, 0.2, 1.0 μg per well of 6-well plate; 1.5 ml culture medium per well). Scale bar, 50 µm. Together, the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis (Appendix Table S16). Subsequently, cholestasis triggered an adaptive response in the liver by down-regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up-regulating the expression of BA efflux transporters Mrp3, Mrp4 and Ostα/β.


Treatment with UDCA ( 13 mg / kg / d ) and GSH ( 40 mg / kg / d ) for 2 weeks restored normal liver function in the child . However , when the treatment was ceased the levels of serum ALT , AST and TBA returned to abnormally high levels in the patient . Interestingly , treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST , and reduced serum TBA in the child patient .

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Treatment with UDCA (13 mg/kg/d) and GSH (40 mg/kg/d) for 2 weeks restored normal liver function in the child. However, when the treatment was ceased the levels of serum ALT, AST and TBA returned to abnormally high levels in the patient. Interestingly, treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST, and reduced serum TBA in the child patient.