( C ) YFP - TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - Fis1 and anti - Cytochrome c antibodies . Scale bars , 20 μm .

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(C) YFP-TBC1D17 together with pcDNA vector or Fis1 was transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-Fis1 and anti-Cytochrome c antibodies. Scale bars, 20 μm.


( D ) YFP or YFP - Fis1 was co - overexpressed with HA - TBC1D15 or HA - TBC1D17 in HEK293 cells . The cell extracts were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) and anti - GFP ( lower panel ) antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YFP or YFP-Fis1 was co-overexpressed with HA-TBC1D15 or HA-TBC1D17 in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.


( E ) YFP , YFP - TBC1D15 , or YFP - TBC1D17 was co - overexpressed with HA - TBC1D15 ( upper panel ) or HA - TBC1D17 ( lower panel ) in HEK293 cells . The cell extracts were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP and anti - HA antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YFP, YFP-TBC1D15, or YFP-TBC1D17 was co-overexpressed with HA-TBC1D15 (upper panel) or HA-TBC1D17 (lower panel) in HEK293 cells. The cell extracts were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP and anti-HA antibodies.


( F ) HA - TBC1D15 ( Δ221 - 250 ) together with Fis1 and YFP - TBC1D15 WT or YFP - TBC1D17 WT were transiently overexpressed ( OE ) in HeLa cells . Cells were subjected to immunostaining with anti - HA and anti - Fis1 antibodies . Images of HA and Fis1 staining were merged in the right panels . Scale bars , 20 μm .

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

(F) HA-TBC1D15 (Δ221-250) together with Fis1 and YFP-TBC1D15 WT or YFP-TBC1D17 WT were transiently overexpressed (OE) in HeLa cells. Cells were subjected to immunostaining with anti-HA and anti-Fis1 antibodies. Images of HA and Fis1 staining were merged in the right panels. Scale bars, 20 μm.


( A ) The indicated cells transiently expressing YFP - GABARAPL1 ( green ) and mCherry - Parkin ( red ) were treated with valinomycin for 3 hr . Scale bars , 10 μm .

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

(A) The indicated cells transiently expressing YFP-GABARAPL1 (green) and mCherry-Parkin (red) were treated with valinomycin for 3 hr. Scale bars, 10 μm.


( B ) YFP - TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST - fused proteins ( GABA , L1 , and L2 represent GABARAP , GABARAPL1 , and GABARAPL2 , respectively ) . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) . Coomassie brilliant blue ( CBB ) staining shows GST - fusion proteins in bound fractions ( lower panel ) . ( C ) Binding assay carried out as in ( B ) with GST - GABARAPL1 WT ( GST - WT ) or its Y49A / L50A mutant ( GST - YL ) . Immunoblotting with anti - GFP antibody ( upper panel ) and CBB staining ( lower panel ) are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YFP-TBC1D15 overexpressed in HEK293 cells was subjected to binding assays with GST-fused proteins (GABA, L1, and L2 represent GABARAP, GABARAPL1, and GABARAPL2, respectively). 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel). Coomassie brilliant blue (CBB) staining shows GST-fusion proteins in bound fractions (lower panel). (C) Binding assay carried out as in (B) with GST-GABARAPL1 WT (GST-WT) or its Y49A/L50A mutant (GST-YL). Immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel) are shown.


( D ) Cell extracts from HEK293 overexpressed HA - TBC1D15 and YFP , YFP - GABARAPL1 ( YFP - WT ) , or its Y49AL50A mutant ( YFP - YL ) were subjected to pull down assays with GFP - Trap . 5 % input and bound fractions were analyzed by immunoblotting with anti - HA ( upper panel ) and anti - GFP ( lower panel ) antibodies .

(D) Cell extracts from HEK293 overexpressed HA-TBC1D15 and YFP, YFP-GABARAPL1 (YFP-WT), or its Y49AL50A mutant (YFP-YL) were subjected to pull down assays with GFP-Trap. 5% input and bound fractions were analyzed by immunoblotting with anti-HA (upper panel) and anti-GFP (lower panel) antibodies.


( E - H ) The indicated YFP - tagged TBC1D15 full - length , truncated , or point - mutant protein or YFP - Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST - GABARAPL1 . 5 % input and bound fractions were analyzed by immunoblotting with anti - GFP antibody ( upper panel ) and CBB staining ( lower panel ) .

(E-H) The indicated YFP-tagged TBC1D15 full-length, truncated, or point-mutant protein or YFP-Fis1 overexpressed in HEK293 cells were subjected to binding assays with recombinant GST-GABARAPL1. 5% input and bound fractions were analyzed by immunoblotting with anti-GFP antibody (upper panel) and CBB staining (lower panel).


( J ) LC3 - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the presence of HA - tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( K ) The YFP - LC3 morphology of cells in ( J ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each well .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LC3-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or F280A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (K) The YFP-LC3 morphology of cells in (J) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each well.


( A ) Total cell lysates from HCT116 treated with control ( NTC ) or RAB7A siRNA were analyzed by immunoblotting .

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

(A) Total cell lysates from HCT116 treated with control (NTC) or RAB7A siRNA were analyzed by immunoblotting.


( B ) The indicated cells stably expressing YFP - LC3 ( green ) and mCherry - Parkin were treated with control ( NTC ) or Rab7 _ # 5 siRNA . After 3 hr valinomycin treatment , cells were analyzed by immunofluorescence microscopy with anti - TOMM20 antibody ( red ) . Z - stacks of confocal images are shown . Magnified images are also shown . Scale bars , 10 μm . ( C ) YFP - LC3 morphologies of cells in ( B ) were quantified . Percentages of cells harboring diffuse , punctuate or accumulated / tubulated YFP - LC3 are shown . Data and error bars were obtained from at least 50 cells in each of three independent replicates .

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

(B) The indicated cells stably expressing YFP-LC3 (green) and mCherry-Parkin were treated with control (NTC) or Rab7_#5 siRNA. After 3 hr valinomycin treatment, cells were analyzed by immunofluorescence microscopy with anti-TOMM20 antibody (red). Z-stacks of confocal images are shown. Magnified images are also shown. Scale bars, 10 μm. (C) YFP-LC3 morphologies of cells in (B) were quantified. Percentages of cells harboring diffuse, punctuate or accumulated/tubulated YFP-LC3 are shown. Data and error bars were obtained from at least 50 cells in each of three independent replicates.


( D ) The indicated cells stably expressing YFP - LC3 ( green ) , mCherry - Parkin , and 2HA - Rab7 ( Red ) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti - HA antibody . Magnified images are also shown . Scale bars , 10 μm .

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

(D) The indicated cells stably expressing YFP-LC3 (green), mCherry-Parkin, and 2HA-Rab7 (Red) were treated with or without valinomycin for 3 hr and analyzed by immunofluorescence microscopy with anti-HA antibody. Magnified images are also shown. Scale bars, 10 μm.


( E ) YFP - LC3 and mCherry - Parkin stably expressing TBC1D15 − / − cells in the presence of HA - tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr . Cells were subjected to immunofluorescence microscopy with anti - HA antibody . Scale bars , 10 μm . ( F ) The YFP - LC3 morphology of cells in ( E ) was quantified . The error bars represent ± SD from three independent replicates . Over 50 cells were counted in each replicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YFP-LC3 and mCherry-Parkin stably expressing TBC1D15−/− cells in the presence of HA-tagged TBC1D15 WT or the D397A mutant were treated with valinomycin for 3 hr. Cells were subjected to immunofluorescence microscopy with anti-HA antibody. Scale bars, 10 μm. (F) The YFP-LC3 morphology of cells in (E) was quantified. The error bars represent ±SD from three independent replicates. Over 50 cells were counted in each replicate.


Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib , Nilotinib , Bosutinib and DMSO ( N = 7 ) . [ Correction added after publication on 4 July 2013 : The y axis of the graph shown in Fig . 1A has been corrected from " concentration ( mM ) " to " concentration ( nM ) " ]

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

Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7). [Correction added after publication on 4 July 2013: The y axis of the graph shown in Fig. 1A has been corrected from "concentration (mM)" to "concentration (nM)"]


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) show total Abl ( top blot ) T412 Abl ( 2nd blot ) , soluble parkin level ( 3rd blot ) , and LC3 ( 4th blot ) relative to MAP - 2 ( N = 9 ) .

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

WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Abl (top blot) T412 Abl (2nd blot), soluble parkin level (3rd blot), and LC3 (4th blot) relative to MAP-2 (N = 9).


Quantitative ELISA showing soluble ( STEN extract ) and insoluble ( 4 M urea extract ) mouseparkin in Tg - APP ( N = 9 ) . Parkin − / − brains were used as a specificity control .

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Quantitative ELISA showing soluble (STEN extract) and insoluble (4 M urea extract) mouseparkin in Tg-APP (N = 9). Parkin−/−brains were used as a specificity control.


( D ) Blots represent immunoprecipitatedBeclin - 1 in mice probed with parkin

(D) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody


( D ) Blots represent immunoprecipitatedBeclin - 1 in mice probed with parkin antibody , and ( E ) control IgG in parallel with immunoprecipitates .

O O 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) Blots represent immunoprecipitatedBeclin-1 in mice probed with parkin antibody, and (E) control IgG in parallel with immunoprecipitates.


( F ) immunoprecipitatedBeclin - 1 probed with parkin and the reverse experiment in post - mortem human AD cortex analyzed on 4 - 12 % SDS - NuPAGE gel .

O O O O O O O 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) immunoprecipitatedBeclin-1 probed with parkin and the reverse experiment in post-mortem human AD cortex analyzed on 4-12% SDS-NuPAGE gel.


In situ proximity ligation assay ( PLA ) shows endogenous parkin - Beclin - 1 complexes in ( G ) WT C57BL / 6mice ( N = 5 ) and ( H ) parkin − / − as control . PLA in Tg - APPmice IP injected once daily for 3 weeks with ( I ) DMSO ( J ) 5 mg / kg Bosutinib and ( K ) 10 mg / kg Nilotinib ( N = 5 ) .

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In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6mice (N = 5) and (H) parkin−/− as control.PLA in Tg-APPmice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5).


PLA in human post - mortem brains in the ( L ) cortex of a normal subject and ( M ) cortex of an AD patient ; the hippocampus of ( N ) a normal subject and ( O ) an AD patient ; the caudate of ( P ) a normal subject and ( Q ) an AD patient .

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

PLA in human post-mortem brains in the (L) cortex of a normal subject and (M) cortex of an AD patient; the hippocampus of (N) a normal subject and (O) an AD patient; the caudate of (P) a normal subject and (Q) an AD patient.


Graph represents human Aβ1 - 42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1 - 42 ( or LacZ ) or Beclin - 1 shRNA for 24 hr , and then treated with 1 μM Bosutinib for an additional 24 hr ( N = 12 ) . * Significantly different to control or as indicated , Mean ± SEM , ANOVA with NeumannKeuls multiple comparison .

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Graph represents human Aβ1-42ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1-42 (or LacZ) or Beclin-1 shRNA for 24 hr, and then treated with 1 μM Bosutinib for an additional 24 hr (N = 12). *Significantly different to control or as indicated, Mean ± SEM, ANOVA with NeumannKeuls multiple comparison.


Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different ( A , B ) Tg - APP + DMSO and ( C ) thioflavin - S staining in Tg - APP treated with DMSO ( N = 7 ) . ( D , E ) Tg - APP treated with 5 mg / kg Bosutinib for 3 - weeks and ( F ) thioflavin - s staining .

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

Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different (A,B) Tg-APP + DMSO and (C) thioflavin-S staining in Tg-APP treated with DMSO (N = 7). (D,E) Tg-APP treated with 5 mg/kg Bosutinib for 3-weeks and (F) thioflavin-s staining.


Staining of 20 μm thick brain sections shows ( G ) parkin , ( H ) Aβ1 - 42 and ( I ) merged figure in cortex of Tg - APPmice after 3 weeks of DMSO treatment , and ( J ) parkin , ( K ) Aβ1 - 42 and ( L ) merged figure in cortex of Tg - APPmice after 3 weeks of 5 mg / kg Bosutinib treatment . ( M ) Parkin , ( N ) Aβ1 - 42 and ( O ) merged figure in hippocampus of Tg - APPmice after 3 weeks of DMSO treatment , and ( P ) parkin , ( Q ) Aβ1 - 42 and ( R ) merged figure in cortex of Tg - APPmice after 3 weeks of Bosutinib treatment ( N = 7 ) .

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

Staining of 20 μm thick brain sections shows (G) parkin, (H) Aβ1-42 and (I) merged figure in cortex of Tg-APPmice after 3 weeks of DMSO treatment, and (J) parkin, (K) Aβ1-42 and (L) merged figure in cortex of Tg-APPmice after 3 weeks of 5 mg/kg Bosutinib treatment. (M) Parkin, (N) Aβ1-42 and (O) merged figure in hippocampus of Tg-APPmice after 3 weeks of DMSO treatment, and (P) parkin, (Q) Aβ1-42 and (R) merged figure in cortex of Tg-APPmice after 3 weeks of Bosutinib treatment (N = 7).


( S ) Graphs represent quantification of amyloidplaques in Tg - APP with and without Bosutinib .

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

(S) Graphs represent quantification of amyloidplaques in Tg-APP with and without Bosutinib.


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing ( T ) levels of BACE1 ( 1st blot ) , ADAM - 10 ( 2nd blot ) and presinilin - 1 ( 3rd blot ) relative to

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

WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (T) levels of BACE1 (1st blot), ADAM-10 (2nd blot) and presinilin-1 (3rd blot) relative to actin


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing ( U ) total APP ( top blot ) , CTFs ( 2nd blot ) and phospho - tyrosine ( 3rd blot ) relative to actin ( N = 9 ) .

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

WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) showing (U) total APP (top blot), CTFs (2nd blot) and phospho-tyrosine (3rd blot) relative to actin (N = 9).


Graphs represent ELISA of human Aβ1 - 42 in ( A ) brain levels and ( B ) bloodAβ1 - 42 levels in 8 - month old Tg - APPmice injected I . P . every other day for 6 weeks with Bosutinib or Nilotinib ( N = 10 ) .

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Graphs represent ELISA of human Aβ1-42 in (A) brain levels and (B) bloodAβ1-42 levels in 8-month old Tg-APPmice injected I.P. every other day for 6 weeks with Bosutinib or Nilotinib (N = 10).


ELISA of human soluble and insoluble brain ( C ) Aβ1 - 42 and ( D ) Aβ1 - 40 levels in 8 - month old Tg - APPmice injected IP daily for 3 weeks with 5 mg / kg Bosutinib ( N = 9 ) .

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ELISA of human soluble and insoluble brain (C) Aβ1-42 and (D) Aβ1-40 levels in 8-month old Tg-APPmice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).


ELISA of ( E ) mouse p - Tau levels in 8 - month old Tg - APP mice injected IP daily for 3 weeks with 5 mg / kg Bosutinib ( N = 9 ) .

O O O O O O 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

ELISA of (E) mouse p-Tau levels in 8-month old Tg-APP mice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).


WB in Tg - APP total brain lysates on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) show total Tau ( 1st blot ) , serine 396 p - Tau ( 2nd blot ) , threonine 231 ( AT180 , 3rd blot ) and serine 199 p - Tau ( AT8 , 4th blot ) relative to actin ( N = 7 ) .

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

WB in Tg-APP total brain lysates on 4-12% NuPAGE SDS gel (Invitrogen) show total Tau (1st blot), serine 396 p-Tau (2nd blot), threonine 231 (AT180, 3rd blot) and serine 199 p-Tau (AT8, 4th blot) relative to actin (N = 7).


Staining of 20μm brain sections shows intracellular Aβ1 - 42 within the ( A ) hippocampus of lentiviralAβ1 - 42 injected WT mice , and ( B ) Bosutinib clearance of intracellular Aβ1 - 42 . Staining of 20μm brain sections shows intracellular Aβ1 - 42 within the ( C ) cortex of WT mice with the lentiviralAβ1 - 42 , and ( D ) Bosutinib clearance of intracellular Aβ1 - 42 ( N = 7 ) .

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

Staining of 20μm brain sections shows intracellular Aβ1-42 within the (A) hippocampus of lentiviralAβ1-42 injected WT mice, and (B) Bosutinib clearance of intracellular Aβ1-42. Staining of 20μm brain sections shows intracellular Aβ1-42 within the (C) cortex of WT mice with the lentiviralAβ1-42, and (D) Bosutinib clearance of intracellular Aβ1-42 (N = 7).


ELISA of human soluble and insoluble Aβ1 - 42 at 6 weeks post - injection of lentiviralAβ1 - 42 and daily treatment with 5 mg / kg Bosutinib ( N = 9 ) for 3 weeks . * Significantly different to control or as indicated , Mean ± SEM , ANOVA with Neumann Keuls multiple comparison .

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

ELISA of human soluble and insoluble Aβ1-42 at 6 weeks post-injection of lentiviralAβ1-42 and daily treatment with 5 mg/kg Bosutinib (N = 9) for 3 weeks. * Significantly different to control or as indicated, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison.


WB on 4 - 12 % NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin − / − mice treated with 5 mg / kg Bosutinib for 3 weeks on 4 - 12 % NuPAGE SDS gel ( Invitrogen ) showing parkin ( 1st blot ) total Abl ( 2nd blot ) , T412 Abl ( 3rd blot ) , Beclin - 1 ( 4th blot ) and LC3 ( 5th blot ) relative to actin ( N = 7 ) .

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

WB on 4-12% NuPAGE SDS gel of total brain lysates in 1 year old wild type and parkin−/−mice treated with 5 mg/kg Bosutinib for 3 weeks on 4-12% NuPAGE SDS gel (Invitrogen) showing parkin (1st blot)total Abl (2nd blot), T412 Abl (3rd blot), Beclin-1 (4th blot) and LC3 (5th blot) relative to actin (N = 7).


Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post - injection with ( G ) lentiviralAβ1 - 42 + 3 weeks DMSO treatment and ( H ) IP injection with 5 mg / kg Bosutinib 3 weeks post - lentiviral expression ( 3 weeks treatment ) clears plaques in WT mice ( N = 7 ) . ( I ) Lentiviral Aβ1 - 42 + DMSO and ( J ) IP injection with 5 mg / kg Bosutinib 3 weeks post - lentiviral expression ( 3 weeks treatment ) in parkin − / − mice ( N = 7 ) .

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

Staining of 20μm brain sections shows plaque formation with 6E10 antibody and DAB 6 weeks post-injection with (G) lentiviralAβ1-42 + 3 weeks DMSO treatment and (H) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) clears plaques in WT mice (N = 7).(I) Lentiviral Aβ1-42 + DMSO and (J) IP injection with 5 mg/kg Bosutinib 3 weeks post-lentiviral expression (3 weeks treatment) in parkin−/−mice (N = 7).


Graphs represent ELISA ( N = 5 ) in autophagic vacuoles ( AVs ) in the brain of 4 and 8 months old Tg - APPmice treated with 10 mg / kg Nilotinib or 5 mg / kg Bosutinib ( N = 5 ) for 3 weeks showing ( A ) humanAβ1 - 42 ( Insert is WB analysis of AVs showing LC3 - B in AV10 and AV20 ( 1st blot ) and LAMP2a in Lys ( 2nd blot ) fraction ) and ( B ) Aβ1 - 40 , ( C ) p - Tau and ( D ) parkin .

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Graphs represent ELISA (N = 5) in autophagic vacuoles (AVs) in the brain of 4 and 8 months old Tg-APPmice treated with 10 mg/kg Nilotinib or 5 mg/kg Bosutinib (N = 5) for 3 weeks showing (A)humanAβ1-42 (Insert is WB analysis of AVs showing LC3-B in AV10 and AV20 (1st blot) and LAMP2a in Lys (2nd blot) fraction) and (B)Aβ1-40,(C) p-Tau and (D) parkin.


ELISA in autophagic vacuoles of 1 year old WT and parkin − / − mice ( N = 5 ) injected with lentiviralAβ1 - 42 for 3 weeks and treated with 10 mg / kg Nilotinib and 5 mg / kg Bosutinib for 3 additional weeks showing ( E ) humanAβ1 - 42 and ( F ) p - Tau levels .

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ELISA in autophagic vacuoles of 1 year old WT and parkin−/−mice (N = 5) injected with lentiviralAβ1-42 for 3 weeks and treated with 10 mg/kg Nilotinib and 5 mg/kg Bosutinib for 3 additional weeks showing (E) humanAβ1-42 and (F) p-Tau levels.


( A ) Represents results of Morris water maze test in lentiviralAβ1 - 42 - injected ± Bosutinib WT ( N = 12 ) and parkin − / − ( N = 10 ) mice , and ( B ) heat maps for each group . Graphs represent total number of entry into platform area and distance travelled .

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(A) Represents results of Morris water maze test in lentiviralAβ1-42-injected ± Bosutinib WT (N = 12) and parkin−/− (N = 10) mice, and (B) heat maps for each group.Graphs represent total number of entry into platform area and distance travelled.


Represents results of Morris water maze test in Tg - APP ± Bosutinib ( N = 12 ) mice . Graphs represent total number of entry into platform area and distance travelled .

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Represents results of Morris water maze test in Tg-APP ± Bosutinib (N = 12) mice.Graphs represent total number of entry into platform area and distance travelled.


( A ) Immuno - precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4 ° C overnight . The control antibody ( ab ) is a rabbit anti - yeast Mad2 .

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(A) Immuno-precipitations of extracts from asynchronous S2 cells using 1 μg of the indicated antibodies at 4°C overnight. The control antibody (ab) is a rabbit anti-yeast Mad2.


( B ) Single confocal section of a population of S2 cells . Examples of interphase cells are boxed in blue . Mitotic cells positive for the anti - phosphoHistone3 ( pH3 ) are boxed in red .

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(B) Single confocal section of a population of S2 cells. Examples of interphase cells are boxed in blue. Mitotic cells positive for the anti-phosphoHistone3 (pH3) are boxed in red.


( C ) Single confocal section of a S2 cell . Anti - Incenp , anti - Cid mark the centromeric chromatin and the inner KT , respectively .

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(C) Single confocal section of a S2 cell. Anti-Incenp, anti-Cid mark the centromeric chromatin and the inner KT, respectively.


( D - E ) Maximal projections of S2 cells imaged by STED microscopy . 7 z - sections for a total of 1 . 5 um ( D ) and 4 z - sections for a total of 0 . 5 um ( E ) are shown . Anti - CenpC and anti - Spc105R mark the proximal and the distal region of different portions of the outer KT , respectively .

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(D-E) Maximal projections of S2 cells imaged by STED microscopy. 7 z-sections for a total of 1.5 um (D) and 4 z-sections for a total of 0.5 um (E) are shown. Anti-CenpC and anti-Spc105R mark the proximal and the distal region of different portions of the outer KT, respectively.


( F - G ) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29 , nuclear pores ( WGA ) , and CyclinB ( F ) , or Snap29 , Spc105R , and the nuclear lamina ( LaminA ) ( G ) . Note the transient perinuclear enrichment of Snap29 in early prophase . WGA stains also cytoplasmic speckles in hemocyte - derived S2 cells ( Rizki Rizki , 1983 ) . High magnification of the insets in C - G shows localization of Snap29 relative to the indicated markers .

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(F-G) Single confocal sections of S2 cells at the indicated mitotic stages stained to detect Snap29, nuclear pores (WGA), and CyclinB (F), or Snap29, Spc105R, and the nuclear lamina (LaminA) (G). Note the transient perinuclear enrichment of Snap29 in early prophase. WGA stains also cytoplasmic speckles in hemocyte-derived S2 cells (Rizki Rizki, 1983). High magnification of the insets in C-G shows localization of Snap29 relative to the indicated markers.


( H - J ) Immuno - gold localization of Snap29 in cryo - sections of S2 cells in pro - metaphase . Pseudo - coloring in red identifies chromosomes ( Ch ) , in yellow membrane organelles and in green MTs . The white arrows in H - I indicate examples of signal in the nucleoplasm that is not associated to membranes , but rather , to electron - dense material . MVB : multivesicular body ; NE : nuclear envelope ; PM : plasma membrane .

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(H-J) Immuno-gold localization of Snap29 in cryo-sections of S2 cells in pro-metaphase. Pseudo-coloring in red identifies chromosomes (Ch), in yellow membrane organelles and in green MTs. The white arrows in H-I indicate examples of signal in the nucleoplasm that is not associated to membranes, but rather, to electron-dense material. MVB: multivesicular body; NE: nuclear envelope; PM: plasma membrane.


( A ) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs . A cartoon of full length Snap29 is shown above the control panel .

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(A) Comparable maximum projections of confocal sections of the dorsal wing pouch portion of third instar larval imaginal discs. A cartoon of full length Snap29 is shown above the control panel.


( B ) Quantification of the Snap29 signal as in panel A , considering > 30 KTs per sample . Relative mRNA expression of the downregulated genes versus control , measured by Q - PCR , is shown above the graph . Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs .

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(B) Quantification of the Snap29 signal as in panel A, considering > 30 KTs per sample. Relative mRNA expression of the downregulated genes versus control, measured by Q-PCR, is shown above the graph. Note that efficient depletion of Zw10 does not affect Snap29 localization to KTs.


( C ) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over - expressed . Cartoons of the expressed Snap29 mutant forms are shown above the panels . Anti - pH3 labels mitotic cells and CenpC marks KTs . ( D ) Quantification of Snap29 as in panel C , considering > 28 KTs per sample . Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells .

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(C) Comparable maximum projections of confocal sections of dorsal wing pouches in which Snap29 has been downregulated and the indicated Snap29 form over-expressed. Cartoons of the expressed Snap29 mutant forms are shown above the panels. Anti-pH3 labels mitotic cells and CenpC marks KTs. (D) Quantification of Snap29 as in panel C, considering > 28 KTs per sample. Note that expression of a form of Snap29 lacking the SNARE1 domain does not rescue Snap29 localization to KTs in Snap29 RNAi cells.


( A ) Selected frames of time - lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29 . Note that depleted cell does not display a recognizable metaphase plate , or fail to segregate all chromosomes correctly , or form tripolar spindles or micronuclei ( arrowheads ; arrowheads in control point to cells dividing normally ) .

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(A) Selected frames of time-lapse imaging of control S2 cells or cells treated for 96 hrs with dsRNA against the central portion of Snap29. Note that depleted cell does not display a recognizable metaphase plate, or fail to segregate all chromosomes correctly, or form tripolar spindles or micronuclei (arrowheads; arrowheads in control point to cells dividing normally).


( B ) Western blotting of extract from control cells and cells depleted of Snap29 ( dsSnap29 ) relative to the experiment in a .

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(B) Western blotting of extract from control cells and cells depleted of Snap29 (dsSnap29) relative to the experiment in a.


( C ) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells , based on time - lapse imaging of an average of > 28 individual cells per sample . P value is determined by two - tailed t - test considering all defects together .

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(C) Quantification of the mitotic phenotype of control and dsSnap29 depleted cells, based on time-lapse imaging of an average of > 28 individual cells per sample. P value is determined by two-tailed t-test considering all defects together.


( D ) Selected frames of time - lapse imaging of control U2OS cells and U2OS cells treated for the indicated time . Note that the depleted cell at 48 - 66h display altered metaphase plate and chromosome segregation ( arrowheads ) .

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(D) Selected frames of time-lapse imaging of control U2OS cells and U2OS cells treated for the indicated time. Note that the depleted cell at 48-66h display altered metaphase plate and chromosome segregation (arrowheads).


( E ) Western blot analysis of extracts relative to the experiments in panel d .

(E) Western blot analysis of extracts relative to the experiments in panel d.


( F ) Quantification of the time from prophase to entry in anaphase based on analysis of > 20 individual cells / sample by time - lapse microscopy . P values are determined by Kruskal - Wallis test with Dunn ' s multiple comparison analysis . OE : Over - expression .

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(F) Quantification of the time from prophase to entry in anaphase based on analysis of >20 individual cells/sample by time-lapse microscopy. P values are determined by Kruskal-Wallis test with Dunn's multiple comparison analysis. OE: Over-expression.


( A ) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4 ° C and immunostained to reveal the presence of unattached kinetochores . CENPT labels all KTs , while MAD1 identifies unattached ones . Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate . ( B ) Quantification of the number of KTs based on attachment , position and MAD1 positivity . Each point per category represents one experiment in which 10 random KTs / cell of 20 cells / sample were counted . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison test . Note that in control cells most of the KT are attached , MAD1 - negative and aligned to the metaphase plate , while in SNAP29KD cells half of the KTs that are unattached , MAD1 - positive and not aligned to the metaphase plate .

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(A) Single confocal sections of HeLa cells at metaphase treated for 8 min at 4°C and immunostained to reveal the presence of unattached kinetochores. CENPT labels all KTs, while MAD1 identifies unattached ones. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate. (B) Quantification of the number of KTs based on attachment, position and MAD1 positivity. Each point per category represents one experiment in which 10 random KTs/cell of 20 cells/sample were counted. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison test. Note that in control cells most of the KT are attached, MAD1-negative and aligned to the metaphase plate, while in SNAP29KD cells half of the KTs that are unattached, MAD1-positive and not aligned to the metaphase plate.


( C ) Single confocal sections of HeLa cells . CREST and CENPT were used to label the centromere region and associated KTs , respectively . Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown . ( D ) Average of the inter KT distance based on in each of which 10 chromosomes / cell and 10 cells / sample were counted . P values are obtained by Kruskal - Wallis test with Dunn ' s multiple comparison analysis . Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs .

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(C) Single confocal sections of HeLa cells. CREST and CENPT were used to label the centromere region and associated KTs, respectively. Enlargements of boxed areas displaying both chromosomes aligned and not aligned at the metaphase plate are shown. (D) Average of the inter KT distance based on in each of which 10 chromosomes/cell and 10 cells/sample were counted. P values are obtained by Kruskal-Wallis test with Dunn's multiple comparison analysis. Note that both aligned and not aligned KTs have a shorter inter KT distance compared to aligned control KTs.


( E ) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA . ( F ) Averaged spindle pole length based on > 20 cells with bipolar spindles / sample . The P value is obtained by two - tailed paired t - test .

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(E) Single confocal sections of HeLa cells at metaphase stained to visualize the mitotic spindle and DNA. (F) Averaged spindle pole length based on >20 cells with bipolar spindles/sample. The P value is obtained by two-tailed paired t-test.


( A - C , E ) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs , released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs .

(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.


( D ) Immunoblotting of HeLa protein extracts , treated as indicated .

(D) Immunoblotting of HeLa protein extracts, treated as indicated.


( F ) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A - C , e relative to CREST . 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining . P values are obtained by 2 - way ANOVA with Sidak ' s multiple comparison analysis relative to localization of HEC1 , which is not lost on SNAP29KD cells .

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(F) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A-C, e relative to CREST. 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Sidak's multiple comparison analysis relative to localization of HEC1, which is not lost on SNAP29KD cells.


( A - C ) KNL1 localization analysis in control , SNAP29 KD and SNAP29 KD expressing full length human SNAP29 , CFP tagged , full length Drosophila Snap29 ( CFP - Snap29 ) , or the mutant forms schematized above the panels . Max projections of single cells in prophase are shown . ( D ) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST . 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells .

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(A-C) KNL1 localization analysis in control, SNAP29 KD and SNAP29 KD expressing full length human SNAP29, CFP tagged, full length Drosophila Snap29 (CFP-Snap29), or the mutant forms schematized above the panels. Max projections of single cells in prophase are shown. (D) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST. 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells.


( E - F ) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs . In cells expressing SNAP29 Q1 Q2 , SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 ( arrows ) .

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(E-F) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs. In cells expressing SNAP29 Q1 Q2, SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 (arrows).


( A - B ) Confocal sections of wild - type and Snap29 mutant eye - antennal imaginal discs ( eye disc portion to the left ) of L3 larvae . Discs were labeled with phalloidin and DAPI to visualize the subcortical f - actin cytoskeleton and nuclei , respectively . Arrowheads in B point to actin - rich unpolarized cells in apical , medial and basal sections of the same disc .

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(A-B) Confocal sections of wild-type and Snap29 mutant eye-antennal imaginal discs (eye disc portion to the left) of L3 larvae. Discs were labeled with phalloidin and DAPI to visualize the subcortical f-actin cytoskeleton and nuclei, respectively. Arrowheads in B point to actin-rich unpolarized cells in apical, medial and basal sections of the same disc.


( C ) High magnification of a single confocal cross - section of wild - type and Snap29 mutant eye disc tissues . Two facing folds of the tissue are separated by the apical lumen . aPKC and Dlg mark the apical and lateral plasma membrane domains , respectively . In Snap29 mutant tissue the apical - basal polarity is disrupted .

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(C) High magnification of a single confocal cross-section of wild-type and Snap29 mutant eye disc tissues. Two facing folds of the tissue are separated by the apical lumen. aPKC and Dlg mark the apical and lateral plasma membrane domains, respectively. In Snap29 mutant tissue the apical-basal polarity is disrupted.


( D ) Single confocal cross - sections of wild - type and Snap29 eye - antennal discs , expressing Puckered - LacZ ( Puc - lacZ ) , a reporter of JNK signaling activation .

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(D) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs, expressing Puckered-LacZ (Puc-lacZ), a reporter of JNK signaling activation.


( E - H ) Single confocal cross - sections of wild - type and Snap29 eye - antennal discs ( E - F ) , and of otherwise wild - type and Snap29 eye - antennal discs expressing the bacterial apoptosis inhibitor p35 ( G - H ) . Anti - activated - Caspase 3 ( Casp3 ) marks apoptotic cells .

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(E-H) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs (E-F), and of otherwise wild-type and Snap29 eye-antennal discs expressing the bacterial apoptosis inhibitor p35 (G-H). Anti-activated-Caspase 3 (Casp3) marks apoptotic cells.


( B ) Representative images of cells released from mitosis for 1 , 2 or 3h in EdU - containing medium . Top , DNA staining with DAPI ; Bottom , EdU immunofluorescence . L , M and H indicate cells with low , medium or high levels of EdU incorporation . The barplot shows the quantification of the proportion of replicating cells at each time point .

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(B) Representative images of cells released from mitosis for 1, 2 or 3h in EdU-containing medium. Top, DNA staining with DAPI; Bottom, EdU immunofluorescence. L, M and H indicate cells with low, medium or high levels of EdU incorporation. The barplot shows the quantification of the proportion of replicating cells at each time point.


Y - axis , z - score ; X - axis , timepoints . A indicates asynchronous cells , M mitotic cells and the numbers represent the minutes after release from the mitotic block ) . Dots show individual replicates and lines averages ( n = 3 for A and M ; n = 2 for each post - mitotic sample ) . Pre - mRNA levels are shown for pluripotency genes ( Oct4 , Klf4 , Esrrb ) and cell cycle regulators ( E2f1 , Cdk2 , Rb ) ; mRNA levels for individual histone genes ( only averages are shown ) .

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Y-axis, z-score; X-axis, timepoints. A indicates asynchronous cells, M mitotic cells and the numbers represent the minutes after release from the mitotic block). Dots show individual replicates and lines averages (n=3 for A and M; n=2 for each post-mitotic sample). Pre-mRNA levels are shown for pluripotency genes (Oct4, Klf4, Esrrb) and cell cycle regulators (E2f1, Cdk2, Rb); mRNA levels for individual histone genes (only averages are shown).


( C ) Percentage of genes presenting at least 1 TF binding site as detected by ChIP - seq within 25kb centred on the TSS , calculated for each gene category shown in ( A ) .

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(C) Percentage of genes presenting at least 1 TF binding site as detected by ChIP-seq within 25kb centred on the TSS, calculated for each gene category shown in (A).


( B ) Heatmaps presented for untreated ( - IAA ; left ) or CTCF - depleted ( + IAA ; right ) CTCF - AID cells .

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(B) Heatmaps presented for untreated (-IAA; left) or CTCF-depleted (+IAA; right) CTCF-AID cells.


( A ) Gene expression changes ( z - score ) in asynchronous cells and during post - mitotic release of CTCF - AID cells treated or not with IAA to deplete CTCF ( blue , presence of CTCF ; red , absence ) . Samples are indicated on the X - axis ( A : asynchronous cells ; M : mitotic cells ; numbers : minutes since release from mitotic block ) . Six gene groups are shown , based on the time of their response to CTCF depletion ( asy , late , early ) and the direction of the transcriptional change ( up , down ) . The number of genes of each group is indicated . The yellow shadow areas indicate times showing expression changes . The boxplots show the median ( bar ) , 25 - 75 % percentiles ( box ) and 1 . 5 - fold inter - quartile range ( whiskers ) .

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(A) Gene expression changes (z-score) in asynchronous cells and during post-mitotic release of CTCF-AID cells treated or not with IAA to deplete CTCF (blue, presence of CTCF; red, absence). Samples are indicated on the X-axis (A: asynchronous cells; M: mitotic cells; numbers: minutes since release from mitotic block). Six gene groups are shown, based on the time of their response to CTCF depletion (asy, late, early) and the direction of the transcriptional change (up, down). The number of genes of each group is indicated. The yellow shadow areas indicate times showing expression changes. The boxplots show the median (bar), 25-75% percentiles (box) and 1.5-fold inter-quartile range (whiskers).


( C ) Average binding profile ( X - axis , reads per million ) of CTCF ( top ) and the Cohesin subunit SMC1 ( bottom ) , across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells ( left ) or either late or early upon mitotic release ( middle and right , respectively ) . Interphase is shown in blue and mitosis in red .

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(C) Average binding profile (X-axis, reads per million) of CTCF (top) and the Cohesin subunit SMC1 (bottom), across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells (left) or either late or early upon mitotic release (middle and right, respectively). Interphase is shown in blue and mitosis in red.


( a ) Nox2 - specific ROS production was assessed using the Nox2 redox biosensor p47 - roGFP redox biosensor Cat : catalase , PEG - Cat : pegylated catalase . (

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(a) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalase. (


b ) Measurement of intracellular glutathione redox potential with Grx1 - roGFP2 .

b) Measurement of intracellular glutathione redox potential with Grx1-roGFP2.


(c) Analysis of Rac1 and (d) Src.


. ( e ) Immunoblot of precipitated p47phox probed with an anti - phosphoserine or anti - p47phox antibody .

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. (e) Immunoblot of precipitated p47phox probed with an anti-phosphoserine or anti-p47phox antibody.


( f ) Nox2 - specific intracellular ROS production was measured using p47 - roGFP redox biosensor .

(f) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor.


g ) Extracellular ROS production was assessed using Amplex - red dye .

g) Extracellular ROS production was assessed using Amplex-red dye.


( h ) Plasma membrane calcium influx was measured by analysing the Fura - 2 fluorescence quench rate upon addition of extracellular Mn2 + .

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(h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+.


( i ) Intracellular RNS generation was measured using DAF - FM . Bars represent average ± s . e . m . from n = 15 individual fibres for each condition in ( a , b , f , g ,

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(i) Intracellular RNS generation was measured using DAF-FM. Bars represent average±s.e.m. from n=15 individual fibres for each condition in (a,b,f,g,i


, j ) . Markers of autophagy were analysed in isolated fibres ( incubated with or without PP2 ) from FDBs .

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,j). Markers of autophagy were analysed in isolated fibres (incubated with or without PP2) from FDBs.


( k ) Autophagosome formation was analysed using fluorescence microscopy ( scale bar = 100 μm ) and illustrated LC3 localization and autophagosome formation . ( l ) Confocal microscopy detected p62 - LC3 localization in single fibres from FDBs ( scale bar = 140 and 50 μm for white box areas ) . All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated . Glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) was detected as a loading control . Representative images are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using analysis of variance ( ANOVA ) with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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(k) Autophagosome formation was analysed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. (l) Confocal microscopy detected p62-LC3 localization in single fibres from FDBs (scale bar=140 and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using analysis of variance (ANOVA) with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) FDB lysates were analysed using immunoblotting with antibodies as indicated .

(a) FDB lysates were analysed using immunoblotting with antibodies as indicated.


( b ) Confocal microscopy shows the effect of Nox2 inhibition on p62 - LC3 localization in single FDB fibres . Representative images of independent biological experiments ( n = 3 ) are shown ( scale bar = 100 and 35 μm for white box areas ) .

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(b) Confocal microscopy shows the effect of Nox2 inhibition on p62-LC3 localization in single FDB fibres. Representative images of independent biological experiments (n=3) are shown (scale bar=100 and 35 μm for white box areas).


( c ) Autophagic flux was analysed by immunoblotting with anti - LC3B and anti - p62 antibodies .

(c) Autophagic flux was analysed by immunoblotting with anti-LC3B and anti-p62 antibodies.


( d ) Effect of gp91 ds and PP2 on LC3 - LAMP1 colocalization was analysed using confocal microscopy . Representative images from independent biological experiments ( n = 3 ) are shown ( scale bar = 140 and 40 μm for red box areas ) .

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(d) Effect of gp91 ds and PP2 on LC3-LAMP1 colocalization was analysed using confocal microscopy. Representative images from independent biological experiments (n=3) are shown (scale bar=140 and 40 μm for red box areas).


(e) Immunoblot analysis of LAMP1,


( g ) Immunoblot of Caspase3 activity . All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated . GAPDH was detected as a loading control . Representative images are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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(g) Immunoblot of Caspase3 activity. All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) Intracellular ROS production was assessed using DCFH - DA . ( b ) Plasma membrane calcium influx was measured by analysing the quench of Fura - 2 fluorescence upon addition of extracellular Mn2 + . ( a , b ) Were conducted in enzymatically digested single FDBs from WT , mdx and p47 − / − - mdx mice . Bars represent average ± s . e . m . from n = 15 individual fibres for each condition .

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(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn2+. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition.


( c ) Immunoblot analysis of Src protein with anti - P - Src or anti - Src antibodies .

(c) Immunoblot analysis of Src protein with anti-P-Src or anti-Src antibodies.


( d ) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated ( c ) and ( d ) were conducted in enzymatically isolated fibres from FDBs of WT , mdx and p47 − / − - mdx mice . Representative images are shown . GAPDH was detected as a loading control . Bars represent average ± s . e . m . from n = 3 independent biological experiments .

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(d) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated (c) and (d) were conducted in enzymatically isolated fibres from FDBs of WT, mdx and p47−/−-mdx mice. Representative images are shown. GAPDH was detected as a loading control. Bars represent average±s.e.m. from n=3 independent biological experiments.


( e ) LC3 - LAMP1 colocalization in single FDBs from mdx and p47 − / − - mdx mice was analysed using confocal microscopy . Representative images from n = 3 independent biological experiments are shown ( scale bar = 100 and 40 μm for red box areas ) .

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(e) LC3-LAMP1 colocalization in single FDBs from mdx and p47−/−-mdx mice was analysed using confocal microscopy. Representative images from n=3 independent biological experiments are shown (scale bar=100 and 40 μm for red box areas).


f ) Immunofluorescence assay to detect lysosomes ( LAMP1 ) in TA muscle from WT , mdx and p47 − / − - mdx mice . White arrows indicate lysosome and yellow arrows indicate nucleus .

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f) Immunofluorescence assay to detect lysosomes (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. White arrows indicate lysosome and yellow arrows indicate nucleus.


( g ) Immunohistochemistry to detect lysosome ( LAMP1 ) in TA muscle from WT , mdx and p47 − / − - mdx mice . Black arrows indicate LAMP1 positive ( brown ) structures . Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre . Representative images from n = 3 independent biological experiments are shown . Scale bar represents 100 μm for f and 140 μm for g . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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(g) Immunohistochemistry to detect lysosome (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. Black arrows indicate LAMP1 positive (brown) structures. Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre. Representative images from n=3 independent biological experiments are shown. Scale bar represents 100 μm for f and 140 μm for g. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( a ) Haematoxylin and eosin staining of cross - sections from Dia showing central nuclei ( arrow head ) and smaller fibres ( arrow ) .

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(a) Haematoxylin and eosin staining of cross-sections from Dia showing central nuclei (arrow head) and smaller fibres (arrow).


( b ) Immunoblot analysis of macrophage content ( CD68 ) .

(b) Immunoblot analysis of macrophage content (CD68).


( c ) Immunofluorescent and bright field images of Dia cross - sections showing fibre - type distribution . Type I ( red ) , IIA ( green ) , IIB / IIX ( white x , unstained and viewed from bright field overlay ) .

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(c) Immunofluorescent and bright field images of Dia cross-sections showing fibre-type distribution. Type I (red), IIA (green), IIB/IIX ( white x, unstained and viewed from bright field overlay).


( d ) Serum creatine kinase activity . ( e ) Force - frequency relationship in Dia muscle strips from WT ( black ) , mdx ( red ) and p47 − / − - mdx ( blue ) mice . Scale bars represent 55 μm . For e , # P0 . 01 p47 − / − - mdx versus mdx . # # P0 . 01 p47 − / − - mdx versus WT and mdx . Mdx was statistically different than WT at all frequencies of stimulation . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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(d) Serum creatine kinase activity. (e) Force-frequency relationship in Dia muscle strips from WT (black), mdx (red) and p47−/−-mdx (blue) mice. Scale bars represent 55 μm. For e, #P0.01 p47−/−-mdx versus mdx.##P0.01 p47−/−-mdx versus WT and mdx. Mdx was statistically different than WT at all frequencies of stimulation. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


( B ) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm .

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(B) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( C ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( B ) , with DAPI staining ( blue ) depicted in addition to PaprikaRFP ( Cas9 ) and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

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(C) Higher magnification of the VZ and SVZ of the electroporated area shown in (B), with DAPI staining (blue) depicted in addition to PaprikaRFP (Cas9) and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( D ) Quantification of the proportion of Cas9 - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid electroporation . Data are the mean of 4 independent experiments ( 7 embryos per condition in total , from 4 litters ) .

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(D) Quantification of the proportion of Cas9-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 plasmid electroporation. Data are the mean of 4 independent experiments (7 embryos per condition in total, from 4 litters).


( F ) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence ( magenta ) showing the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm .

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(F) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence (magenta) showing the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.


( G ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( F ) , with DAPI staining ( blue ) depicted in addition to mCherry and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

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(G) Higher magnification of the VZ and SVZ of the electroporated area shown in (F), with DAPI staining (blue) depicted in addition to mCherry and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


H ) Quantification of the proportion of mCherry - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 protein electroporation . Data are the mean of 4 independent experiments ( 5 embryos per condition in total , from 4 litters ) .

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H) Quantification of the proportion of mCherry-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 protein electroporation. Data are the mean of 4 independent experiments (5 embryos per condition in total, from 4 litters).


( I ) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

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(I) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


( J ) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence ( magenta ) and the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm .

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(J) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence (magenta) and the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.


K ) Quantification of the proportion of Cas9 - positive cells ( left ) and mCherry - positive cells ( right ) in the VZ plus SVZ that are GFP - positive 24 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid ( left ) or protein ( right ) electroporation . Data are the mean of 3 independent experiments each ( 3 embryos per condition in total , from 3 litters ) . ( D , H , K ) Controls were set to 100 % and the gGFP conditions expressed relative to control ( D , 11 % ; H , 24 % ; K left , 40 % K right , 28 % ) . Error bars indicate SD ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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K) Quantification of the proportion of Cas9-positive cells (left) and mCherry-positive cells (right) in the VZ plus SVZ that are GFP-positive 24 h after control (Con, white) or gGFP (black) Cas9 plasmid (left) or protein (right) electroporation. Data are the mean of 3 independent experiments each (3 embryos per condition in total, from 3 litters).(D, H, K) Controls were set to 100% and the gGFP conditions expressed relative to control (D, 11%; H, 24%; K left, 40% K right, 28%). Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.


( B ) Daughter cells of single aRGCs microinjected with either Cas9 / control gRNA ( top ) or Cas9 / gGFP ( bottom ) revealed by Dx - A555immunofluorescence ( magenta ) ; Cells 1 , aRGC daughter ; Cells 2 , BP daughter . Dashed lines indicate ventricular surface . Images are maximum intensity projections of 20 ( Control ) and 24 ( gGFP ) optical sections . Scale bars , 20 µm .

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(B) Daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555immunofluorescence (magenta); Cells 1, aRGC daughter; Cells 2, BP daughter. Dashed lines indicate ventricular surface. Images are maximum intensity projections of 20 (Control) and 24 (gGFP) optical sections. Scale bars, 20 µm.


( C ) Single optical sections of Cells 1 and Cells 2 depicted in ( B ) , showing the effects of Cas9 and control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( as revealed by immunofluorescence , green ) . Cells 1 and Cells 2 are indicated by dotted lines ; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9 / gGFP microinjection . Scale bars , 5 µm .

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(C) Single optical sections of Cells 1 and Cells 2 depicted in (B), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression (as revealed by immunofluorescence, green). Cells 1 and Cells 2 are indicated by dotted lines; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9/gGFP microinjection. Scale bars, 5 µm.


( D ) Quantification of the proportion of daughter cells ( Dx - A555 + ) of microinjected cells that show GFP expression 24 h after control ( Con , white ) or gGFP ( black ) microinjection . Control is set to 100 % and the gGFP condition expressed relative to control ( 39 % ) . Data are the mean of two independent experiments ( 3 embryos and 1 litter per experiment , total number of daughter cells scored : control 142 , gGFP 88 ) ; bars indicate the variation of the two individual values from the mean ( P < 0 . 05 ) .

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(D) Quantification of the proportion of daughter cells (Dx-A555+) of microinjected cells that show GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. Control is set to 100% and the gGFP condition expressed relative to control (39%). Data are the mean of two independent experiments (3 embryos and 1 litter per experiment, total number of daughter cells scored: control 142, gGFP 88); bars indicate the variation of the two individual values from the mean (P<0.05).


( A - D ) Neocortex of mouseE13 . 5embryos was in utero electroporated with a plasmid encoding , under constitutive promoters , Cas9 _ T2A _ PaprikaRFP and gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( Tbr2 ) , followed by analysis at E15 . 5 . ( A ) Electroporated area showing the effects of Cas9 expression ( revealed by PaprikaRFPDAPI , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on aRGCs expression ( green , immunofluorescence ) ; blue , Cas9staining . Boxes indicate areas shown at higher magnification in the insets ( 45 x 45 μm ) . Dotted lines indicate nuclei of progeny of electroporated Cas9 ; note the presence of Tbr2 immunoreactivity in the control ( top ) and its absence upon Cas9 / gTbr2 plasmid electroporation ( bottom ) . Scale bars , 20 μm .

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(A-D) Neocortex of mouseE13.5embryos was in utero electroporated with a plasmid encoding, under constitutive promoters, Cas9_T2A_PaprikaRFP and gRNA targeting either LacZ (Control, Con) or Tbr2 (Tbr2), followed by analysis at E15.5.(A) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFPDAPI, magenta) together with either control gRNA (top) or gTbr2 (bottom) on aRGCs expression (green, immunofluorescence); blue, Cas9staining. Boxes indicate areas shown at higher magnification in the insets (45 x 45 μm). Dotted lines indicate nuclei of progeny of electroporated Cas9; note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 plasmid electroporation (bottom). Scale bars, 20 μm.


( B ) Quantification of the proportion of Cas9 + cells that are Tbr2 + 48 h after control ( Con , white ) or gTbr2 ( black ) plasmid electroporation . Data are the mean of 4 independent experiments ( 6 embryos per condition , from 4 litters ) .

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(B) Quantification of the proportion of Cas9+ cells that are Tbr2+ 48 h after control (Con, white) or gTbr2 (black) plasmid electroporation. Data are the mean of 4 independent experiments (6 embryos per condition, from 4 litters).


( C ) Electroporated area showing the effects of Cas9 expression ( revealed by PaprikaRFP fluorescence , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on the abundance of mitotic progenitors as revealed by phosphohistone H3 ( PH3 ) immunofluorescence ( green ) . Apical and basal mitoses are indicated by arrows and arrowheads , respectively ; note the reduction in mitotic Cas9 + BPs upon Cas9 / gTbr2 plasmid electroporation . Scale bars , 20 μm .

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(C) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFP fluorescence, magenta) together with either control gRNA (top) or gTbr2 (bottom) on the abundance of mitotic progenitors as revealed by phosphohistone H3 (PH3) immunofluorescence (green). Apical and basal mitoses are indicated by arrows and arrowheads, respectively; note the reduction in mitotic Cas9+ BPs upon Cas9/gTbr2 plasmid electroporation. Scale bars, 20 μm.


( D ) Quantification of apical and basal Cas9 + mitoses ( as revealed by PH3 immunofluorescence ) in a unit area 48 h after Cas9 / gTbr2 plasmid electroporation . Data are expressed as percentage of the data obtained upon control plasmid electroporation and are the mean of 3 independent experiments ( 3 embryos per condition in total , from 3 litters ) ; note the reduction in basal ( black , 68 % ) but not apical ( white ) mitoses .

(D) Quantification of apical and basal Cas9+ mitoses (as revealed by PH3 immunofluorescence) in a unit area 48 h after Cas9/gTbr2 plasmid electroporation. Data are expressed as percentage of the data obtained upon control plasmid electroporation and are the mean of 3 independent experiments (3 embryos per condition in total, from 3 litters); note the reduction in basal (black, 68%) but not apical (white) mitoses.


( E - H ) Single aRGCs in neocortex in organotypic slices of telencephalon of E14 . 5 mouse embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( gTbr2 ) and with Dextran 10 , 000 - Alexa 555 ( Dx - A555 ) , followed by analysis after 48 h of culture . ( E ) Progeny of single microinjected aRGCs ( revealed by Dx - A555immunofluorescence , magenta , dotted lines ) showing the effects of Cas9 together with either control gRNA ( top ) or gTbr2 ( bottom ) on Tbr2 expression ( green , immunofluorescence ) ; blue , DAPI staining . Note the presence of Tbr2 immunoreactivity in the control ( top ) and its absence upon Cas9 / gTbr2 ( bottom ) protein microinjection . Images are single optical sections . Scale bars , 5 µm .

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(E-H) Single aRGCs in neocortex in organotypic slices of telencephalon of E14.5 mouse embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ (Control, Con) or Tbr2 (gTbr2) and with Dextran 10,000-Alexa 555 (Dx-A555), followed by analysis after 48 h of culture.(E) Progeny of single microinjected aRGCs (revealed by Dx-A555immunofluorescence, magenta, dotted lines) showing the effects of Cas9 together with either control gRNA (top) or gTbr2 (bottom) on Tbr2 expression (green, immunofluorescence); blue, DAPI staining. Note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 (bottom) protein microinjection. Images are single optical sections. Scale bars, 5 µm.


( F ) Quantification of the proportion of the progeny ( Dx - A555 + ) of microinjected aRGCs that show Tbr2 expression 48 h after control ( Con , white ) or gTbr2 ( black ) microinjection . Data are the mean of 3 independent experiments ( 3 embryos per condition ; total number of cells scored : control 45 , gTbr2 41 ) .

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(F) Quantification of the proportion of the progeny (Dx-A555+) of microinjected aRGCs that show Tbr2 expression 48 h after control (Con, white) or gTbr2 (black) microinjection. Data are the mean of 3 independent experiments (3 embryos per condition; total number of cells scored: control 45, gTbr2 41).


( G ) Distribution across the cortical wall of the progeny ( revealed by Dx - A555immunofluorescence , magenta ; arrows , aRGC ; arrowheads , BPs ; open arrow , neuron ) of single control ( left ) or gTbr2 ( right ) microinjected aRGCs . Dashed lines indicate ventricular surface ( bottom ) and basal lamina ( top ) . Images are maximum intensity projections of 67 ( Control ) and 94 ( gTbr2 ) single optical sections . Scale bars , 20 µm .

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(G) Distribution across the cortical wall of the progeny (revealed by Dx-A555immunofluorescence, magenta; arrows, aRGC; arrowheads, BPs; open arrow, neuron) of single control (left) or gTbr2 (right) microinjected aRGCs. Dashed lines indicate ventricular surface (bottom) and basal lamina (top). Images are maximum intensity projections of 67 (Control) and 94 (gTbr2) single optical sections. Scale bars, 20 µm.


( H ) Quantification of the distribution of the progeny ( Dx - A555 + ) of microinjected aRGCs in the VZ , SVZ and intermediate zone / cortical plate ( CP + IZ ) 48 h after control ( Con , white ) or gTbr2 ( black ) microinjection ( total number of cells scored : control 37 , gTbr2 51 , pooled from at least 3 independent experiments ) . Note that upon gTbr2 microinjection the distribution of cells across the various cortical zones is significantly different ( VZ : control 77 % , gTbr2 39 : SVZ : control 20 % , gTbr2 37 % ; IZ / CP : control 3 % , gTbr2 24 % , * , P < 0 . 05 ) . ( B , F ) Controls were set to 100 % and the gTbr2 conditions expressed relative to control ( B , 48 % ; F , 50 % ) . ( B , D , F ) Error bars indicate SD ( B , D , F ) ; * , P < 0 . 05 ; * * , P < 0 . 01 .

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(H) Quantification of the distribution of the progeny (Dx-A555+) of microinjected aRGCs in the VZ, SVZ and intermediate zone/cortical plate (CP+IZ) 48 h after control (Con, white) or gTbr2 (black) microinjection (total number of cells scored: control 37, gTbr2 51, pooled from at least 3 independent experiments). Note that upon gTbr2 microinjection the distribution of cells across the various cortical zones is significantly different (VZ: control 77%, gTbr2 39: SVZ: control 20%, gTbr2 37%; IZ/CP: control 3%, gTbr2 24%, *, P<0.05).(B, F) Controls were set to 100% and the gTbr2 conditions expressed relative to control (B, 48%; F, 50%).(B, D, F) Error bars indicate SD (B, D, F); *, P<0.05; **, P<0.01.


Figure 4 . Sequencing analysis of the CRISPR / Cas9 - induced disruption of the Tbr2 locus . ( A - C ) Next - generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off - target loci generated from PaprikaRFP - expressing cells isolated at E15 . 5 from neocortex of mouse embryos in utero electroporated at E13 . 5 with plasmids encoding Cas9 _ T2A _ PaprikaRFP and either gTbr2 ( black ) or control gRNA ( Con , white ) as in Fig 3 A - D . ( A ) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off - target sites ( OT1 - 4 ) . Data are expressed as percentage of the total number of reads and are the mean of two independent experiments ( 8 and 5 embryos per condition , respectively ) . ( B , C ) Analysis of one of the experiments of ( A ) for deletions ( B ) and insertions ( C ) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9 / gTbr2 plasmid electroporation .

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Figure 4. Sequencing analysis of the CRISPR/Cas9-induced disruption of the Tbr2 locus.(A-C) Next-generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off-target loci generated from PaprikaRFP-expressing cells isolated at E15.5 from neocortex of mouse embryos in utero electroporated at E13.5 with plasmids encoding Cas9_T2A_PaprikaRFP and either gTbr2 (black) or control gRNA (Con, white) as in Fig 3 A-D.(A) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off-target sites (OT1-4). Data are expressed as percentage of the total number of reads and are the mean of two independent experiments (8 and 5 embryos per condition, respectively).(B, C) Analysis of one of the experiments of (A) for deletions (B) and insertions (C) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9/gTbr2 plasmid electroporation.


C Genomic PCR analysis indicating the existence of MpFGMYB in both male [ Y ] and female [ X ] genomes of M . polymorpha . Two biological replicates were analyzed . Constitutively expressed MpEF1α was used as a control .

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C Genomic PCR analysis indicating the existence of MpFGMYB in both male [Y] and female [X] genomes of M. polymorpha. Two biological replicates were analyzed. Constitutively expressed MpEF1α was used as a control.


D Real - time RT - PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes . MpEF1α was used for normalization . Measurements of six biological replicates for thalli and sporophyte , and three biological replicates for gametangiophore are plotted . Bars represent mean ± SD . Symbols above the bars indicate grouping by p < 0 . 05 in a Tukey - Kramer test .

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D Real-time RT-PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes. MpEF1α was used for normalization. Measurements of six biological replicates for thalli and sporophyte, and three biological replicates for gametangiophore are plotted. Bars represent mean ± SD. Symbols above the bars indicate grouping by p <0.05 in a Tukey-Kramer test.


E A transcriptional reporter with 5 ′ - and 3 ′ - flanking sequences revealed transcription of MpFGMYB throughout mature archegonia . Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine fluorescence .

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E A transcriptional reporter with 5′- and 3′-flanking sequences revealed transcription of MpFGMYB throughout mature archegonia. Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.


F MpFGMYB - Citrine fusion proteins expressed using the 5 ′ - and 3 ′ - flanking sequences rescued the Mpfgmybge - 1 mutant and accumulated in the nuclei of the egg and the ventral canal cell ( VCC ) Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine fluorescence .

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F MpFGMYB-Citrine fusion proteins expressed using the 5′- and 3′-flanking sequences rescued the Mpfgmybge-1 mutant and accumulated in the nuclei of the egg and the ventral canal cell (VCC) Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.


G A transcriptional AtMYB64 reporter ( AtMYB64 - NYG ) is specifically expressed in all four cell types of the A . thaliana embryo sac Scale bar : 25 µm . Green , YFP fluorescence ; white , cell walls .

G A transcriptional AtMYB64 reporter (AtMYB64-NYG) is specifically expressed in all four cell types of the A. thaliana embryo sac Scale bar: 25 µm. Green, YFP fluorescence; white, cell walls.


H Expression of AtMYB64 - Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells ( CC ) and egg cells ( EC ) of the A . thaliana embryo sac Scale bar : 25 µm . Green , Citrine fluorescence ; white , cell walls .

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H Expression of AtMYB64-Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells (CC) and egg cells (EC) of the A. thaliana embryo sac Scale bar: 25 µm. Green, Citrine fluorescence; white, cell walls.


B Diagnosis of genetic sex using Y - and X - chromosome - linked rbm27 and rhf73 markers , respectively . Two biological replicates were analyzed for each genotype .

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B Diagnosis of genetic sex using Y- and X-chromosome-linked rbm27 and rhf73 markers, respectively. Two biological replicates were analyzed for each genotype.


C - L Gametangiophore morphology ( C - G ) and gamete development ( H - L ) of wild - type and mutant plants . Scale bars , 5 mm ( C - G ) , 10 µm ( H and J ) , 100 µm ( I , K , and L ) .

C-L Gametangiophore morphology (C-G) and gamete development (H-L) of wild-type and mutant plants. Scale bars, 5 mm (C-G), 10 µm (H and J), 100 µm (I, K, and L).


A - C DAPI - staining visualization of sperm formation in wild - type male ( A ) , Mpfgmyb [ X ] ( B ) , and Mpfgmyb [ Y ] ( C ) plants . Note that background DAPI staining visualizes flagella ( arrows ) in addition to nuclei ( arrowheads ) . ( B ′ ) is an enlarged image of the boxed region in ( B ) , visualizing an incompletely condensed nucleus . Scale bars , 5 µm .

A-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bars, 5 µm.


D RT - PCR analysis indicating acquisition of male - like autosomal gene expression patterns in Mpfgmyb [ X ] antheridiophores . Two independent Mpfgmyb [ X ] mutant alleles were analyzed .

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D RT-PCR analysis indicating acquisition of male-like autosomal gene expression patterns in Mpfgmyb [X] antheridiophores. Two independent Mpfgmyb [X] mutant alleles were analyzed.


E , F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [ X ] sperm ( F ) , as compared with those of wild - type males ( E ) . Scale bars , 100 nm .

E, F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [X] sperm (F), as compared with those of wild-type males (E). Scale bars, 100 nm.


A RNA - seq analysis showing male - specific accumulation of lncRNAs derived from the MpFGMYB 3 ′ region . Diagrams illustrate wild - type and mutant MpFGMYB / SUF loci . Folded lines with a ∆ symbol indicate a deletion .

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A RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region. Diagrams illustrate wild-type and mutant MpFGMYB/SUF loci. Folded lines with a ∆ symbol indicate a deletion.


B RT - PCR analysis of wild - type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far - red irradiation . Two independent suf mutant alleles were analyzed . The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms .

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B RT-PCR analysis of wild-type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far-red irradiation. Two independent suf mutant alleles were analyzed. The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms.


C - H Gametangiophore morphology ( C , E and G ) and gametangium development ( D , F and H ) of plants with the designated genotypes . Scale bars , 1 mm ( C , E and G ) , 20 µm ( D ) , 50 µm ( F ) , 100 µm ( H ) .

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C-H Gametangiophore morphology (C, E and G) and gametangium development (D, F and H) of plants with the designated genotypes. Scale bars, 1 mm (C, E and G), 20 µm (D), 50 µm (F), 100 µm (H).


A gametangiophore of MpEF1pro : SUF / suf - 30ge [ Y ] , indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [ Y ] . Scale bar , 2mm .

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A gametangiophore of MpEF1pro:SUF/suf-30ge [Y], indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [Y]. Scale bar, 2mm.


B Real - time RT - PCR analyses confirming SUF transcript accumulation in SUF - overexpressing lines . Constitutively expressed MpEF1α was used as control . Measurements of six biological replicates for WT [ Y ] , and three biological replicates for each SUF - overexpressing line are plotted . Data information : bars represent mean ± SD . Asterisks indicate significant differences from WT [ Y ] ( B ) ( p < 0 . 05 , two - tailed Student ' s t - test ) .

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B Real-time RT-PCR analyses confirming SUF transcript accumulation in SUF-overexpressing lines. Constitutively expressed MpEF1α was used as control. Measurements of six biological replicates for WT [Y], and three biological replicates for each SUF-overexpressing line are plotted. Data information: bars represent mean ± SD. Asterisks indicate significant differences from WT [Y] (B) (p <0.05, two-tailed Student's t-test).