Figure 2N2a 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2N2a 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 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).


Figure 3 ( 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 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 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 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 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 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 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).


Figure 4 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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).(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).


Figure 5 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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).(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).


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 µm


Figure 7 ( 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 ) . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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).(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.(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).


Figure 1Results of three independent microscopy - based screens in U2OS cells . Knockdown of Luciferase ( blue ) and the positive controls PPM1D and βTrCP ( red ) are indicated . A low / high Z - score indicates less / more phospho - histone H3 positive cells compared to the control , reflecting reduced / increased numbers of cells going into mitosis in the respective knockdowns after 5 Gy of IR . Deconvolution of the 41 primary hits in U2OS cells by four separate siRNA oligonucleotides . Grey represents a recovery value that is similar to the Luciferase control ( < 0 . 5 SD ) and the red represents an altered recovery value ( > 0 . 5 SD ) in two independent experiments . U2OS cells ( D ) and RPE1 hTert immortalized primary epithelial cells ( E ) were transfected with siRNA oligos targeting the hits in a similar setup as described in A ) , but now analyzed for mitotic entry by MPM2 and PI staining by flow cytometry . For normalization see materials and methods . Error bars represent the SEM of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Results of three independent microscopy-based screens in U2OS cells. Knockdown of Luciferase (blue) and the positive controls PPM1D and βTrCP (red) are indicated. A low/high Z-score indicates less/more phospho-histone H3 positive cells compared to the control, reflecting reduced/increased numbers of cells going into mitosis in the respective knockdowns after 5 Gy of IR. Deconvolution of the 41 primary hits in U2OS cells by four separate siRNA oligonucleotides. Grey represents a recovery value that is similar to the Luciferase control (<0.5 SD) and the red represents an altered recovery value (>0.5 SD) in two independent experiments.U2OS cells (D) and RPE1 hTert immortalized primary epithelial cells (E) were transfected with siRNA oligos targeting the hits in a similar setup as described in A), but now analyzed for mitotic entry by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.


Figure 2RPE1 cells were depleted for p53 in combination with the indicated proteins by siRNA , in conditions used for the primary screen ( 2 Gy of IR ) . Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry . For normalization see materials and methods . Error bars represent the SEM of three independent experiments . U2OS cells were transfected with the indicated siRNA oligonucleotides in a setup similar to the primary screen , but with 10 Gy of IR and in the presence of ATM and ATR inhibitors . Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry . For normalization see materials and methods . Error bars represent the SEM of three independent experiments . U2OS cells were transfected with the indicated siRNA oligos using the protocol of the primary screen and fixed for immunofluorescence after 0 , 2 and 24 h after 5 Gy IR . Samples were stained with antibodies for γH2AX ( D ) or 53BP1 ( E ) and analyzed through automated confocal microscopy and specifically developed image analysis software . From one experiment two to four separate images with a total of at least 100 cells were analyzed for each knockdown . Shown are the number of γH2AX ( D ) and 53BP1 ( E ) foci per cell . In the box plots , the median with the first and third quartile is indicated , and the whiskers are drawn down to the 10th percentile and up to the 90th .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2RPE1 cells were depleted for p53 in combination with the indicated proteins by siRNA, in conditions used for the primary screen (2 Gy of IR). Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.U2OS cells were transfected with the indicated siRNA oligonucleotides in a setup similar to the primary screen, but with 10 Gy of IR and in the presence of ATM and ATR inhibitors. Mitotic entry was analyzed by MPM2 and PI staining by flow cytometry. For normalization see materials and methods. Error bars represent the SEM of three independent experiments.U2OS cells were transfected with the indicated siRNA oligos using the protocol of the primary screen and fixed for immunofluorescence after 0, 2 and 24 h after 5 Gy IR. Samples were stained with antibodies for γH2AX (D) or 53BP1 (E) and analyzed through automated confocal microscopy and specifically developed image analysis software. From one experiment two to four separate images with a total of at least 100 cells were analyzed for each knockdown. Shown are the number of γH2AX (D) and 53BP1 (E) foci per cell. In the box plots, the median with the first and third quartile is indicated, and the whiskers are drawn down to the 10th percentile and up to the 90th.


Figure 3U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6 , synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls . U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6 , synchronized in G2 or cells were lysed and analyzed using western blotting with the indicated antibodiesU2OS WT , PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP - PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 stainingU2OS WT , PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP - PHF6 were synchronized in G2 cells were lysed and analyzed using western blotting with the indicated antibodiesU2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence . In ( F ) is presented 53BP1 IRIF intensity . In ( G ) is shown the number of γH2AX foci per cell . U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis . The comet tail moment was analyzed in at least 50 individual cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 and subsequent recovery after 5 Gy of IR and the addition of nocodazole was analyzed by flow cytometry with MPM2 staining PPM1D and βTrCP were used as positive controls.U2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2 or cells were lysed and analyzed using western blotting with the indicated antibodiesU2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 stainingU2OS WT, PHF6 knockout and PHF6 knock out cells that were reconstituted with GFP-PHF6 were synchronized in G2 cells were lysed and analyzed using western blotting with the indicated antibodiesU2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR and cells were fixed at different time points for immunofluorescence. In (F) is presented 53BP1 IRIF intensity. In (G) is shown the number of γH2AX foci per cell.U2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis. The comet tail moment was analyzed in at least 50 individual cells.


Figure 4U2OS cells stably expressing GFP - PHF6 and mCherry - NBS1 were laser - irradiated and cells were analyzed by time - lapse imaging . U2OS cells stably expressing GFP - PHF6 were laser - irradiated and cells were analyzed by time - lapse imaging . In ( B ) , the PARP inhibitor Olaparib was added 30 min before laser - induced damage . At least 60 cells were analyzed in three individual experiments and error bars represent the SD ( lower panel ) . Scale bar is 5 μM . GC92 SV40 immortalized human fibroblasts containing the I - SceI NHEJ reporter were transfected with the indicated siRNA oligos and I - SceI . 48 h later the cells were fixed and analyzed by flow cytometry . Represented is the relative repair efficiency as compared to the Luciferase control . Clonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2 , 3 or 4 Gy IR . Shown is the relative survival as compared to the undamaged control . Error bars represent the SEM of three individual experiments . GC92 SV40 immortalized human fibroblasts containing the I - SceI NHEJ reporter were transfected with the indicated siRNA oligos and I - SceI . 48 h later genomic DNA was extracted and repair of I - SceI cut - sites analyzed through junction analysis ( n = independently derived sequences , see materials and methods for details ) .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4U2OS cells stably expressing GFP-PHF6 and mCherry-NBS1 were laser-irradiated and cells were analyzed by time-lapse imaging.U2OS cells stably expressing GFP-PHF6 were laser-irradiated and cells were analyzed by time-lapse imaging. In (B), the PARP inhibitor Olaparib was added 30 min before laser-induced damage. At least 60 cells were analyzed in three individual experiments and error bars represent the SD (lower panel). Scale bar is 5 μM.GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later the cells were fixed and analyzed by flow cytometry. Represented is the relative repair efficiency as compared to the Luciferase control.Clonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2, 3 or 4 Gy IR. Shown is the relative survival as compared to the undamaged control. Error bars represent the SEM of three individual experiments.GC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later genomic DNA was extracted and repair of I-SceI cut-sites analyzed through junction analysis (n= independently derived sequences, see materials and methods for details).


Figure 1 ( B ) NSC - 34 and SH - SY5Y cells were transfected with various concentrations of siPRPH or siNTC . The efficiency of siRNA knockdown at 48h . p . t . was validated by Western blot while cytotoxicity was determined by AlamarBlue assay . siPRPH - or siNTC - transfected cells were infected with EV - A71 S41 , and the viral titers were determined at 48h . p . i .

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Figure 1(B) NSC-34 and SH-SY5Y cells were transfected with various concentrations of siPRPH or siNTC. The efficiency of siRNA knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay. siPRPH- or siNTC-transfected cells were infected with EV-A71 S41, and the viral titers were determined at 48h.p.i.


Figure 2Two - week old AG129 mice ( n = 2 - 3 / group / time point ) were infected with EV - A71 S41 . ( A ) At various time points post - infection , mice were euthanized and the relative expression of PRPH was detected by Western blot in tissue homogenates ( brainstem , spinal cord and limb muscle ) . Actin and GAPDH serve as loading controls , and protein expression was compared to age - matched non - infected mice . Two - week old AG129 mice ( n = 2 - 3 / group / time point ) were infected with EV - A71 S41 . ( B ) Immunostaining of PRPH and EV - A71 particles on brainstem and spinal cord sections harvested at 6 d . p . i .

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Figure 2Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (A) At various time points post-infection, mice were euthanized and the relative expression of PRPH was detected by Western blot in tissue homogenates (brainstem, spinal cord and limb muscle). Actin and GAPDH serve as loading controls, and protein expression was compared to age-matched non-infected mice.Two-week old AG129 mice (n=2-3 /group/time point) were infected with EV-A71 S41. (B) Immunostaining of PRPH and EV-A71 particles on brainstem and spinal cord sections harvested at 6 d.p.i.


Figure 3 ( C ) Proximity ligation assay . NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 10 and 0 . 01 , respectively . Cells were fixed at 48h . p . i . prior to PLA staining using anti - PRPH and anti - VP0 antibodies .

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Figure 3(C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. Cells were fixed at 48h.p.i. prior to PLA staining using anti-PRPH and anti-VP0 antibodies.


Figure 4 ( A ) NSC - 34 and SH - SY5Y cells were reverse - transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV - A71 RNA . The culture supernatants were harvested for viral titer determination at 24h . p . t .

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Figure 4(A) NSC-34 and SH-SY5Y cells were reverse-transfected with siPRPH or siNTC for 48 hrs prior to transfection with EV-A71 RNA. The culture supernatants were harvested for viral titer determination at 24h.p.t.


Figure 5 ( A ) NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 30 and 0 . 01 , respectively . At 48 h . p . i . , cells were fixed and permeabilized prior to immunostaining . ( B ) Total infected cell lysates were pulled down with anti - PRPH or IgG isotype control antibody prior to immunoblotting . Uninfected cells were included as negative control . IP : immunoprecipitation ; IB : immunoblotting . C ) Proximity ligation assay . NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 10 and 0 . 01 , respectively . At 48 h . p . i . , the cells were fixed and permeabilized prior to PLA staining using antibodies specific to PPRH , VP0 , 3C and 3D .

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Figure 5(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48 h.p.i., cells were fixed and permeabilized prior to immunostaining.(B) Total infected cell lysates were pulled down with anti-PRPH or IgG isotype control antibody prior to immunoblotting. Uninfected cells were included as negative control. IP: immunoprecipitation; IB: immunoblotting.C) Proximity ligation assay. NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 10 and 0.01, respectively. At 48 h.p.i., the cells were fixed and permeabilized prior to PLA staining using antibodies specific to PPRH, VP0, 3C and 3D.


Figure 6 ( C ) Viral titers in siRNA - treated NSC - 34 cells . Cells were transfected with various concentrations of siRNA specific to the indicated target genes . The efficiency of siRNA - mediated knockdown at 48h . p . t . was validated by Western blot while cytotoxicity was determined by AlamarBlue assay siRNA - or siNTC - transfected cells were infected with EV - A71 S41 at MOI 10 , and the viral titers were determined at 48h . p . i . ( D ) Drug treatment . EV - A71 - infected NSC - 34 cells were treated with the indicated drugs , and the viral titers were determined at 48h . p . i .

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Figure 6(C) Viral titers in siRNA-treated NSC-34 cells. Cells were transfected with various concentrations of siRNA specific to the indicated target genes. The efficiency of siRNA-mediated knockdown at 48h.p.t. was validated by Western blot while cytotoxicity was determined by AlamarBlue assay siRNA- or siNTC-transfected cells were infected with EV-A71 S41 at MOI 10, and the viral titers were determined at 48h.p.i.(D) Drug treatment. EV-A71-infected NSC-34 cells were treated with the indicated drugs, and the viral titers were determined at 48h.p.i.


Figure 7 ( A ) NSC - 34 and SH - SY5Y cells were infected with EV - A71 S41 at MOI 30 and 0 . 01 , respectively . At 48h . p . i . , the cells were fixed and permeabilized prior to immunostaining . B ) Proximity ligation assay . NSC - 34 and SH - SY5Y cells were transfected with LucEV - A71 replicon . Cells were fixed and permeabilized at 24h . p . t . prior to PLA staining using antibodies specific to Rac1 , 3C and 3D .

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Figure 7(A) NSC-34 and SH-SY5Y cells were infected with EV-A71 S41 at MOI 30 and 0.01, respectively. At 48h.p.i., the cells were fixed and permeabilized prior to immunostaining.B) Proximity ligation assay. NSC-34 and SH-SY5Y cells were transfected with LucEV-A71 replicon. Cells were fixed and permeabilized at 24h.p.t. prior to PLA staining using antibodies specific to Rac1, 3C and 3D.


Figure 1Representative composite immune - fluorescent images of whole hair follicle units from non - lesional and lesional scalp psoriasis patients . K15 ( green ) , c - JUN ( red A , C ) and JUNB ( red B , D ) and DAPI ( blue ) . Confocal images of the bulge region of human psoriatic hair follicles from non - lesional and lesional regions of the scalp . K15 ( green ) , c - JUN ( red E , G ) and JUNB ( red F , H ) and DAPI ( blue ) . Percentage of HF - SCs ( K15 + ) that express c - JUN ( I ) and JUNB ( J ) in the bulge region of human psoriatic hair follicles from non - lesional and lesional scalp in comparison with healthy scalp . n = 2 - 6 hair follicles in anagen per group from five psoriatic patients and two healthy patients .

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Figure 1Representative composite immune-fluorescent images of whole hair follicle units from non-lesional and lesional scalp psoriasis patients. K15 (green), c-JUN (red A, C) and JUNB (red B, D) and DAPI (blue).Confocal images of the bulge region of human psoriatic hair follicles from non-lesional and lesional regions of the scalp. K15 (green), c-JUN (red E, G) and JUNB (red F, H) and DAPI (blue).Percentage of HF-SCs (K15+) that express c-JUN (I) and JUNB (J) in the bulge region of human psoriatic hair follicles from non-lesional and lesional scalp in comparison with healthy scalp. n=2-6 hair follicles in anagen per group from five psoriatic patients and two healthy patients.


Figure 2Composite immunofluorescent images showing GFP expression of whole - ear sections from DKO * - mT / mG mice at day 5 , 7 , 15 and 30 . Increased GFP expression is shown at day 7 followed by progressive decrease of GFP + keratinocytes ( white dotted line represents basal layer , and red dotted line represents outermost skin layer ) . n = 3 per time point . Quantification analysis of GFP expression ( top ) and epidermal thickness ( bottom ) of DKO * mice at different time points during psoriasis - like disease progression . n = 6 per time point . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to control group ) . Quantification analysis of cleaved Caspase - 3 ( cCas3 top ) and Ki67 ( bottom ) in ear skin of DKO * mice at different time points during psoriasis - like disease progression . n = 3 per time point . Intravital confocal time - lapse imaging of non - mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO * - mT / mG at day 7 after first tamoxifen injection . Dotted circles emphasize areas in which mutant GFP + keratinocytes are replaced by non - mutant Tomato + keratinocytes . Percentage of GFP + area eliminated during intravital confocal time - lapse imaging at day 5 , 7 and 15 in DKO * - mT / mG mice ( Time - lapse 1 : 8h ) . Positive value : Area of GFP increased . Negative value : Area of GFP reduced . Fluorescence imaging of DKO * - mT / mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP + keratinocytes reside along the hair follicles . White dotted line separates epidermis and dermis . White arrows represent GFP + hair follicles . Intravital confocal time - lapse imaging of DKO * - mT / mG at day 15 after first tamoxifen injection . Mutant GFP + Keratinocytes ( white arrows ) are maintained around hair follicles .

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Figure 2Composite immunofluorescent images showing GFP expression of whole-ear sections from DKO*-mT/mG mice at day 5, 7, 15 and 30. Increased GFP expression is shown at day 7 followed by progressive decrease of GFP+ keratinocytes (white dotted line represents basal layer, and red dotted line represents outermost skin layer). n=3 per time point. Quantification analysis of GFP expression (top) and epidermal thickness (bottom) of DKO* mice at different time points during psoriasis-like disease progression. n=6 per time point. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).Quantification analysis of cleaved Caspase-3 (cCas3 top) and Ki67 (bottom) in ear skin of DKO* mice at different time points during psoriasis-like disease progression. n=3 per time point.Intravital confocal time-lapse imaging of non-mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO*-mT/mG at day 7 after first tamoxifen injection. Dotted circles emphasize areas in which mutant GFP+ keratinocytes are replaced by non-mutant Tomato+ keratinocytes.Percentage of GFP+ area eliminated during intravital confocal time-lapse imaging at day 5, 7 and 15 in DKO*-mT/mG mice (Time-lapse 1:8h). Positive value: Area of GFP increased. Negative value: Area of GFP reduced.Fluorescence imaging of DKO*-mT/mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP+ keratinocytes reside along the hair follicles. White dotted line separates epidermis and dermis. White arrows represent GFP+ hair follicles.Intravital confocal time-lapse imaging of DKO*-mT/mG at day 15 after first tamoxifen injection. Mutant GFP+ Keratinocytes (white arrows) are maintained around hair follicles.


Figure 3Ear pictures of control , DKO * , and DKO * K15 mice after 15 days of induction show clear signs of inflammation and psoriatic - like scales in both models . Representative images of hematoxylin and eosin ( H & E ) staining of ear sections from control , DKO * and DKO * K15 mice . Ear thickness measurement at different time points during psoriasis - like development in control , DKO * and DKO * K15 mice . n > 5 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to control group ) . FACS quantification of CD45 + cells from ear skin of control , DKO * and DKO * K15 mice at different time points during psoriasis - like development . n > 5 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to controls ) . Quantification of IL - 17A , IL - 6 and LCN2 production in sera of control , DKO * and DKO * K15 mice at day 30 of psoriasis - like disease by ELISA . n > 4 per group . Statistical significance * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - student two tailed - test relative to controls ) . Quantification of total number of GFP + epidermal cells by FACS analysis from DKO * - mT / mG and DKO * K15 - mT / mG mice at different time points during psoriasis - like progression compared to Co - mT / mG mice . n = 3 - 6 per group and time point . Representative immunofluorescence images of ear sections from CoK15 - mT / mG and DKO * K15 - mT / mG mouse models after 15 days of induction . Red ( Tomato ) , green ( GFP ) and blue ( DAPI ) . Hyperplasia of Tomato + keratinocytes in psoriatic - like DKO * K15 - mT / mG mouse with clusters of GFP + keratinocytes are derived from hair follicle K15 + GFP + stem cells . n = 3 per group . Dotted lines separate epidermis and dermis . Quantification analysis of cleaved Caspase - 3 ( cCas3 ) and Ki67 in ear skin of DKO * K15 - mT / mG mice at different time points during psoriasis - like disease progression .

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Figure 3Ear pictures of control, DKO*, and DKO*K15 mice after 15 days of induction show clear signs of inflammation and psoriatic-like scales in both models.Representative images of hematoxylin and eosin (H&E) staining of ear sections from control, DKO* and DKO*K15 mice.Ear thickness measurement at different time points during psoriasis-like development in control, DKO* and DKO*K15 mice. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).FACS quantification of CD45+ cells from ear skin of control, DKO* and DKO*K15 mice at different time points during psoriasis-like development. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).Quantification of IL-17A, IL-6 and LCN2 production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n > 4 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).Quantification of total number of GFP+ epidermal cells by FACS analysis from DKO*-mT/mG and DKO*K15-mT/mG mice at different time points during psoriasis-like progression compared to Co-mT/mG mice. n = 3-6 per group and time point.Representative immunofluorescence images of ear sections from CoK15-mT/mG and DKO*K15-mT/mG mouse models after 15 days of induction. Red (Tomato), green (GFP) and blue (DAPI). Hyperplasia of Tomato+ keratinocytes in psoriatic-like DKO*K15-mT/mG mouse with clusters of GFP+ keratinocytes are derived from hair follicle K15+ GFP+ stem cells. n=3 per group. Dotted lines separate epidermis and dermis. Quantification analysis of cleaved Caspase-3 (cCas3) and Ki67 in ear skin of DKO*K15-mT/mG mice at different time points during psoriasis-like disease progression.


Figure 4Average number of live and dead ( TOPRO - 3 + Dapi + ) primary keratinocytes derived from HF - SCGFP at different time points of time lapse capture during 48 hours . There is a slight increase over time of live HF - SCs from DKO * mice when compared to control cells , and no difference in average of dead cells . n = 4 fields from two independent experiments . Average number of live and dead ( TOPRO - 3 + DAPI + ) primary keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO * basal live keratinocytes at 12 hours , followed by a significant increase in cell death at 48 hours . Average number of live primary keratinocytes from co - cultures of GFP + with Tomato + bulge HF - SCs at different time points of time lapse capture during 48 hours . Co - culture of HF - SCGFP with HF - SCTom from DKO * - mT / mG mice shows a significant increase of HF - SCTom , while the HF - SCTom from DKO * - mT / mG mice show a normal growth when they are co - culture with HF - SCGFP from control mice . Average number of live primary keratinocytes from co - cultures of GFP + with Tomato + basal keratinocytes at different time points of time lapse capture during 48 hours . Co - culture of b - KCGFP with b - KCTom from DKO * - mT / mG mice show significant increase of b - KCTom , while these b - KCTom from DKO * - mT / mG mice show a normal growth when they are co - culture with b - KCGFP from control mice . Average total number of live Tomato + basal keratinocytes ( from DKO * or control mice ) at different time points cultured in the bottom part of trans - well system cultures with GFP + basal keratinocytes ( from DKO * or Control mice ) cultured in the upper trans - well ( Tw ) . n = 5 fields from three independent experiments . Tom + basal keratinocytes significantly increased when these were cultured with mutant GFP + basal keratinocytes in the trans - well in comparison with control GFP + basal keratinocytes .

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Figure 4Average number of live and dead (TOPRO-3+ Dapi+) primary keratinocytes derived from HF-SCGFP at different time points of time lapse capture during 48 hours. There is a slight increase over time of live HF-SCs from DKO* mice when compared to control cells, and no difference in average of dead cells. n = 4 fields from two independent experiments. Average number of live and dead (TOPRO-3+ DAPI+) primary keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO* basal live keratinocytes at 12 hours, followed by a significant increase in cell death at 48 hours.Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ bulge HF-SCs at different time points of time lapse capture during 48 hours. Co-culture of HF-SCGFP with HF-SCTom from DKO*-mT/mG mice shows a significant increase of HF-SCTom, while the HF-SCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with HF-SCGFP from control mice. Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ basal keratinocytes at different time points of time lapse capture during 48 hours. Co-culture of b-KCGFP with b-KCTom from DKO*-mT/mG mice show significant increase of b-KCTom, while these b-KCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with b-KCGFP from control mice.Average total number of live Tomato+ basal keratinocytes (from DKO* or control mice) at different time points cultured in the bottom part of trans-well system cultures with GFP+ basal keratinocytes (from DKO* or Control mice) cultured in the upper trans-well (Tw). n = 5 fields from three independent experiments. Tom+ basal keratinocytes significantly increased when these were cultured with mutant GFP+ basal keratinocytes in the trans-well in comparison with control GFP+ basal keratinocytes.


Figure 5Heat map for specific genes relative to biological process enriched based on Gene Ontology IDs for DEGs that are : i ) exclusively up - regulated in HF - SCGFP ; ii ) exclusively up - regulated in HF - SCTom ; iii ) commonly up - regulated in HF - SCGFP and HF - SCTom . Gene expression of TSLP in different epidermal cell subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice . protein expression of TSLP in different epidermal cell subpopulations sorted at mid - term ( D7 ) or late - term ( D30 ) of psoriasis - like progression in DKO * mice . n = 2 - 3 . Quantification of TSLP production in sera of control , DKO * and DKO * K15 mice at day 30 of psoriasis - like disease by ELISA . n = 4 per group .

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Figure 5Heat map for specific genes relative to biological process enriched based on Gene Ontology IDs for DEGs that are: i) exclusively up-regulated in HF-SCGFP; ii) exclusively up-regulated in HF-SCTom; iii) commonly up-regulated in HF-SCGFP and HF-SCTom.Gene expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice.protein expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice. n=2-3.Quantification of TSLP production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n = 4 per group.


Figure 6TSLP production in conditioned media quantified by ELISA three days after infection with Ad - empty or Ad - cre . n = 3 independent experiments . Percentage of EdU + Tomato + keratinocytes after blocking with anti - TSLP . Anti - TSLP neutralizes the hyper - proliferation of non - mutantTom KCs in vitro after infection with Ad - cre . Gene expression of Tslpr after TSLP neutralization is reduced . Gene expression of IL - 7ra after TSLP neutralization is increased . Gene expression of VEGFα after TSLP neutralization is reduced . Representative images of treated ears after 9 days of anti - TSLP or IgG treatment . Longitudinal analysis of ear thickness in DKO * mice at different time points during psoriasis - like disease progressionPercentage of EdU + epidermal cells ( CD45 - CD49f + ) after blocking with anti - TSLP in DKO * and DKO * 15 miceRepresentative immune - fluorescence images of ear sections from control or DKO * mice treated with IgG or anti - TSLP ) stained for Ki67 . Ki67 expression was reduced in mice treated with anti - TSLP ( n = 3 ) .

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Figure 6TSLP production in conditioned media quantified by ELISA three days after infection with Ad-empty or Ad-cre. n=3 independent experiments.Percentage of EdU+ Tomato+ keratinocytes after blocking with anti-TSLP. Anti-TSLP neutralizes the hyper-proliferation of non-mutantTom KCs in vitro after infection with Ad-cre.Gene expression of Tslpr after TSLP neutralization is reduced. Gene expression of IL-7ra after TSLP neutralization is increased. Gene expression of VEGFα after TSLP neutralization is reduced.Representative images of treated ears after 9 days of anti-TSLP or IgG treatment.Longitudinal analysis of ear thickness in DKO* mice at different time points during psoriasis-like disease progressionPercentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 miceRepresentative immune-fluorescence images of ear sections from control or DKO* mice treated with IgG or anti-TSLP) stained for Ki67. Ki67 expression was reduced in mice treated with anti-TSLP (n =3).


Figure 7Representative composite immune - fluorescent images of whole hair follicle units by confocal from non - lesional and lesional scalp psoriasis patients . c - JUN ( purple ) , K - 15 ( green ) , TSLP ( red ) and DAPI ( blue ) . TSLP intensity measurement in hair follicle units and epidermis by ImageJ from healthy scalp , non - lesional and lesional scalp psoriasis patients . Representation of fold change relative to control healthy scalp . n = 3 samples . Representative composite immune - fluorescent images of whole hair follicle units by confocal from non - lesional and lesional scalp psoriasis patients ( D ) and magnification images of specific regions from the HFs ( E ) . CD200 ( green ) , c - JUN ( white ) , TSLP ( red ) , DAPI ( blue ) .

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Figure 7Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients. c-JUN (purple), K-15 (green), TSLP (red) and DAPI (blue).TSLP intensity measurement in hair follicle units and epidermis by ImageJ from healthy scalp, non-lesional and lesional scalp psoriasis patients. Representation of fold change relative to control healthy scalp. n= 3 samples.Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients (D) and magnification images of specific regions from the HFs (E). CD200 (green), c-JUN (white), TSLP (red), DAPI (blue).


Figure 1B Spermatocyte chromosomes that underwent condensation ( right ) within a MII oocyte ( MII chromosome mass , left ) . In this study , spermatocytes picked up for injection had tetrad chromosomes ready for condensation . C Chromosomal analysis of MII oocytes that had been injected with primary spermatocytes . In the spermatocyte - injected groups , normality was improved by reducing the ooplasm mass ( * P < 0 . 005 by Fisher ' s exact probability test ) . Arrows in the right figure indicate prematurely separated chromatids . The numbers of oocytes observed are indicated on the top of the bars .

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Figure 1B Spermatocyte chromosomes that underwent condensation (right) within a MII oocyte (MII chromosome mass, left). In this study, spermatocytes picked up for injection had tetrad chromosomes ready for condensation. C Chromosomal analysis of MII oocytes that had been injected with primary spermatocytes. In the spermatocyte-injected groups, normality was improved by reducing the ooplasm mass (*P < 0.005 by Fisher's exact probability test). Arrows in the right figure indicate prematurely separated chromatids. The numbers of oocytes observed are indicated on the top of the bars.


Figure 2B Chromosome tracking in 3D . The reconstructed images are viewed from the side of the metaphase plate . Signals are interpolated in the Z axis for visualization . White and red arrowheads , as well as red surfaces , indicate univalent - like chromosomes that underwent unbalanced predivision ( premature segregation of sister chromatids ) . Scale bar = 4 μm . C Halving the recipient ooplasmic mass rescued chromosome alignment . The numbers of misaligned chromosomes and their parental origin were determined in 3D ( n = 39 and 17 oocytes ) . Error bars show the standard deviation . Student ' s t - test was used to compare means . * P < 0 . 05 . N . S . , not significant .

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Figure 2B Chromosome tracking in 3D. The reconstructed images are viewed from the side of the metaphase plate. Signals are interpolated in the Z axis for visualization. White and red arrowheads, as well as red surfaces, indicate univalent-like chromosomes that underwent unbalanced predivision (premature segregation of sister chromatids). Scale bar = 4 μm. C Halving the recipient ooplasmic mass rescued chromosome alignment. The numbers of misaligned chromosomes and their parental origin were determined in 3D (n = 39 and 17 oocytes). Error bars show the standard deviation. Student's t-test was used to compare means. *P < 0.05. N.S., not significant.


Figure 3C Univalent - like chromosomes . Images were 3D - reconstructed and viewed from the top of the metaphase plate . Red surfaces with white arrowheads indicate univalent - like chromosomes . Scale bar = 3 μm . D Halving the ooplasm volume suppressed the premature separation of paternal chromosomes . Oocytes were categorized based on whether the chromosomes exhibited premature separation into univalent - like structures prior to segregation errors . Data from 39 and 17 oocytes , respectively .

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Figure 3C Univalent-like chromosomes. Images were 3D-reconstructed and viewed from the top of the metaphase plate. Red surfaces with white arrowheads indicate univalent-like chromosomes. Scale bar = 3 μm.D Halving the ooplasm volume suppressed the premature separation of paternal chromosomes. Oocytes were categorized based on whether the chromosomes exhibited premature separation into univalent-like structures prior to segregation errors. Data from 39 and 17 oocytes, respectively.


Figure 4D Effect of forced SAC activation . Oocytes expressing MAD1 - mNeonGreen - CENP - C ( green ) , together with major satellite - mClover ( green ) and H2B - mCherry ( red ) , were imaged . Time after the start of imaging ( h : mm ) is shown . Oocytes were categorized based on anaphase figures ( n = 24 , 23 oocytes ) .

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Figure 4D Effect of forced SAC activation. Oocytes expressing MAD1-mNeonGreen-CENP-C (green), together with major satellite-mClover (green) and H2B-mCherry (red), were imaged. Time after the start of imaging (h:mm) is shown. Oocytes were categorized based on anaphase figures (n = 24, 23 oocytes).


Figure 1GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( A ) Representative maximum intensity projection images of 10 Z - stacks per image . Scale bar : 10 µm . GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( B ) Quantification of GFP foci per 0 . 0025 mm2 . Values are presented as mean ± standard deviation of the mean and were calculated from at least three independent experiments with 3 individuals per replicate . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to the T - test . GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( C ) GFP - ATG8a cleavage immunoblot for plants exposed to the same treatments as in ( A ) . Numbers below the blots represent ratio for given sample normalized to input and relative to non - treated control . Experiments were repeated minimum 3 times with similar results . GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( D ) NBR1 immunoblot for atg2 - 2 samples for given treatments . Numbers below the blots represent ratio for given sample normalized to input and relative to non - treated control . Experiments were repeated minimum 3 times with similar results .

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Figure 1GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (A) Representative maximum intensity projection images of 10 Z-stacks per image. Scale bar: 10 µm.GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (B) Quantification of GFP foci per 0.0025 mm2. Values are presented as mean ± standard deviation of the mean and were calculated from at least three independent experiments with 3 individuals per replicate. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (C) GFP-ATG8a cleavage immunoblot for plants exposed to the same treatments as in (A). Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (D) NBR1 immunoblot for atg2-2 samples for given treatments. Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.


Figure 2 ( A ) Seedlings were acclimated for 16 h in MS containing ABA and then imaged 30 min after being swapped to MS containing ABA / NAA ( control ) , MS or MS containing flg22 . Images are representative maximum intensity projection of 10 Z - stacks per condition . Experiments were repeated 3 times independently with similar results . Scale bar : 10 µm . ( B ) Quantification of GFP foci per 0 . 0025 mm2 , for samples treated as described in A . Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments , with 3 individuals per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to the T - test . ( C ) GFP - ATG8a cleavage immunoblot for given treatments . The ratio of free GFP to loading control , normalized to the 16h pre - treated sample ( set to 1 ) is provided below each band . ( D ) Seedlings were acclimated for 16 h in MS containing NAA and then imaged 30 min after being swapped to MS containing ABA / NAA ( control ) , MS or MS containing 6 - BA . Images are representative maximum intensity projection of 10 Z - stacks per condition . Experiments were repeated 3 times independently with similar results . Scale bar : 10 µm . ( E ) Quantification of GFP foci per 0 . 0025 mm2 , for samples treated as described in ( D ) . Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments , with 3 individuals per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to the T - test . ( F ) GFP - ATG8a cleavage immunoblot for given treatments . The ratio of free GFP to loading control , normalized to the 16h pre - treated sample ( set to 1 ) is provided below each band .

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Figure 2(A) Seedlings were acclimated for 16 h in MS containing ABA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing flg22. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (B) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in A. Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. (C) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.(D) Seedlings were acclimated for 16 h in MS containing NAA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing 6-BA. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (E) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in (D). Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. (F) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.


Figure 3 ( B ) Pattern correlation used to find proteins that accumulate upon ABA / NAA treatment and are removed in WT but not in atg2 after swapping to flg22 / 6 - BA . Protein clusters obtained after quantitative proteomics of WT ( green ) and atg2 - 2 ( magenta ) samples treated as described ( C ) Protein cluster fitting the pattern displayed ( D ) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with flg22 . Protein clusters obtained after quantitative proteomics of WT ( green ) and atg2 - 2 ( magenta ) samples treated as described ( E ) Protein cluster of proteins which accumulate upon NAA treatment and are removed in WT but not in atg2 after swapping to 6 - BA ( F ) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with 6 - BA .

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Figure 3(B) Pattern correlation used to find proteins that accumulate upon ABA/NAA treatment and are removed in WT but not in atg2 after swapping to flg22/6-BA.Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described (C) Protein cluster fitting the pattern displayed (D) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with flg22.Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described (E) Protein cluster of proteins which accumulate upon NAA treatment and are removed in WT but not in atg2 after swapping to 6-BA (F) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with 6-BA.


Figure 4 ( B ) Dry weight of WT , atg5 - 1 or atg2 - 2 plants grown under stable ( 21 / 16 ºC 16 / 8 - h photoperiod ) or following the Swedish Spring of 2013 . Box plots : centerlines show the medians ; box limits indicate the 25th and 75th percentiles ; whiskers extend to the minimum and maximum . Significance calculated by Kruskal - Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods . Asterisks represent significant pairwise differences ( ∗ ∗ ∗ ∗ P < 0 . 0001 ) ( C ) Box plots displaying the heterogeneity of samples in ( B ) . Box plots : centerlines show the medians ; box limits indicate the 25th and 75th percentiles ; whiskers extend to the minimum and maximum . Significance calculated by Kruskal - Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods . Asterisks represent significant pairwise differences ( ∗ ∗ P < 0 . 01 , ∗ ∗ ∗ ∗ P < 0 . 0001 )

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Figure 4(B) Dry weight of WT, atg5-1 or atg2-2 plants grown under stable (21/16 ºC 16/8-h photoperiod) or following the Swedish Spring of 2013. Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods. Asterisks represent significant pairwise differences ( ∗∗∗∗P< 0.0001) (C) Box plots displaying the heterogeneity of samples in (B). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods. Asterisks represent significant pairwise differences (∗∗P < 0.01, ∗∗∗∗P< 0.0001)


Figure 5 ( A ) Representative images of root explants from WT , atg2 - 2 and atg5 - 1 in CIM ( 6 days ) or CIM + SIM ( 6 + 21 days ) . Scale bar : 2 cm ( B ) Quantification ( % ) of explants presenting shoots after 21 days after incubation on SIM . Results were obtained from 3 independent experiments with at least 20 calli per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to the T - test .

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Figure 5(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (6 days) or CIM + SIM (6 + 21 days). Scale bar: 2 cm(B) Quantification (%) of explants presenting shoots after 21 days after incubation on SIM. Results were obtained from 3 independent experiments with at least 20 calli per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


Figure 6 ( A ) Representative images of root explants from WT , atg2 - 2 and atg5 - 1 in CIM ( 21 days ) or CIM + SIM ( 21 + 21 days or 21 + 35 days ) . Scale bar : 2 cm ( B and C ) Fresh weights of calli after incubation on CIM ( B ) or CIM + SIM ( 21 + 21 ) ( C ) . Box plots : centerlines show the medians ; box limits indicate the 25th and 75th percentiles ; whiskers extend to the minimum and maximum . Results were obtained from 3 independent experiments with at least 20 calli per condition , asteriscs mark statistical significance to WT according to the T - test ( * * P < 0 . 001 ; * * * P < 0 . 0001 ) .

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Figure 6(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (21 days) or CIM + SIM (21+ 21 days or 21+ 35 days). Scale bar: 2 cm(B and C) Fresh weights of calli after incubation on CIM (B) or CIM + SIM (21+ 21) (C). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Results were obtained from 3 independent experiments with at least 20 calli per condition, asteriscs mark statistical significance to WT according to the T -test (** P<0.001; *** P<0.0001).


Figure 1Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1 . Western Blot of Chk1 in U2OS cells , 48 h after transfection with siRNA . Actin was used as loading control . Control ( siLuc ) and Chk1 - depleted ( siChk1 ) U20S cells were labeled with CldU and IdU for 10 and 30 min , respectively . Mean ( + SEM ) track lengths ( top ) and CldU / IdU ratios ( bottom ) are shown . Data come from 6 independent experiments and a total of 579 ( siLuc ) and 590 ( siChk1 ) fibers were scored . Western Blot of phospho - serine - 296 - Chk1 ( p - Chk1 ) in U2OS cells . Cells were treated 1 . 5 h with the Chk1 inhibitor Gö6976 ( Chk1i ) or DMSO , irradiated with 100J / m2 UV to induce high levels of p - Chk1 , and collected 1 . 5 h afterwards . Actin was used as a loading control . U2OS cells treated with DMSO ( control ) or Chk1i were labeled with CldU and IdU for 10 and 30 min , respectively . Data come from 3 independent experiments and a total of 304 ( DMSO ) and 312 ( Chk1i ) fibers were scored .

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Figure 1Labelling scheme and representative DNA fibers from U2OS cells transfected with siLuc or siChk1.Western Blot of Chk1 in U2OS cells, 48 h after transfection with siRNA. Actin was used as loading control.Control (siLuc) and Chk1-depleted (siChk1) U20S cells were labeled with CldU and IdU for 10 and 30 min, respectively. Mean (+SEM) track lengths (top) and CldU/IdU ratios (bottom) are shown. Data come from 6 independent experiments and a total of 579 (siLuc) and 590 (siChk1) fibers were scored.Western Blot of phospho-serine-296-Chk1 (p-Chk1) in U2OS cells. Cells were treated 1.5 h with the Chk1 inhibitor Gö6976 (Chk1i) or DMSO, irradiated with 100J/m2 UV to induce high levels of p-Chk1, and collected 1.5 h afterwards. Actin was used as a loading control.U2OS cells treated with DMSO (control) or Chk1i were labeled with CldU and IdU for 10 and 30 min, respectively. Data come from 3 independent experiments and a total of 304 (DMSO) and 312 (Chk1i) fibers were scored.