F . Representative Western blots of total palmitoylated DLK ( upper panel ) of DLK palmitoylation ( lower panel ) from cultured DRG neurons subjected to 3h of NGF deprivation ( n = 5 independent experiments ; Mann - Whitney U ) . Data information : Graphs represent mean ± in ( F ) . * p < 0 . 05 * * p < 0 . 01 .

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F. Representative Western blots of total and palmitoylated DLK (upper panel) and quantification of DLK palmitoylation levels (lower panel) from cultured DRG neurons subjected to 3h of NGF deprivation (n = 5 independent experiments; Mann-Whitney U test). Data information: Graphs represent mean ± SEM in (F). *p < 0.05 and **p < 0.01.


A . Representative stills from time - lapse recordings of DIV6 DRG neurons overexpressing DLK - GFP . Arrowheads point to moving DLK - GFP positive structures . B . Kymographs made from complete time - lapse recording shown in A . left shows original kymograph , right shows an illustration of manually traced structure displacements for better visualization . Data information : Scale bar represents 10 μm in ( B ) 5 μm in ( A )

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative stills from time-lapse recordings of DIV6 DRG neurons overexpressing DLK-GFP. Arrowheads point to individual moving DLK-GFP positive structures. B. Kymographs made from the complete time-lapse recording shown in A. The left shows the original kymograph, and the right shows an illustration of the manually traced structure displacements for better visualization. Data information: Scale bar represents 10 μm in (B) 5 μm in (A)


C . Representative of DIV3 DRG neurons nucleofected with a non - targeting siRNA ( NT siRNA ) or a siRNA against DLK ( DLK siRNA ) , stained for endogenous DLK β3 - tubulin ( TUBB3 ) . On right , of DLK mean in neurons expressing control NT or DLK siRNAs ( n = 25 neurons / condition ; Unpaired t ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 10 μm in ( C ) 2 μm in zooms in ( C ) .

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C. Representative images of DIV3 DRG neurons nucleofected with a non-targeting siRNA (NT siRNA) or a siRNA against DLK (DLK siRNA), and stained for endogenous DLK and β3-tubulin (TUBB3). On the right, quantification of the DLK mean intensity in neurons expressing control NT or DLK siRNAs (n = 25 neurons/condition; Unpaired t test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (C) 2 μm in zooms in (C).


D - F . Representative kymographs ( D ) , of GFP - positive particle number ( E ) of anterograde , retrograde , reverse or non - moving GFP - positive particles ( F ) from DLK - GFP time - lapse recordings in DIV6 DRG neurons , in control situation ( + NGF ) or after 3h of NGF deprivation ( - NGF ) ( n = 45 - 46 neurons / condition ; Mann - Whitney U in E Chi - square in F ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents , 5 μm in ( D )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Representative kymographs (D), quantification of GFP-positive particle number (E) and percentage of anterograde, retrograde, reverse or non-moving GFP-positive particles (F) from DLK-GFP time-lapse recordings in DIV6 DRG neurons, in control situation (+ NGF) or after 3h of NGF deprivation (-NGF) (n = 45-46 neurons/ condition; Mann-Whitney U test in E and Chi-square test in F). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents , 5 μm in (D)


G . Representative of DIV3 cultured embryonic DRG neurons maintained in presence ( + NGF ) or absence ( - NGF ) of NGF for 3h stained for endogenous DLK . On right , of number of DLK - positive in control situation or after 3h of NGF deprivation ( n = 18 - 19 neurons / condition ; Mann - Whitney U ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 5 μm in ( G )

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

G. Representative images of DIV3 cultured embryonic DRG neurons maintained in the presence (+NGF) or absence (-NGF) of NGF for 3h and stained for endogenous DLK. On the right, quantification of the number of DLK- positive puncta in control situation or after 3h of NGF deprivation (n = 18-19 neurons/condition; Mann-Whitney U test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 5 μm in (G)


H . Representative kymograph stills from a time lapse recording of 6DIV neurons overexpressing a GFP - tagged N - terminal region of DLK ( NtermDLK - GFP ) . Arrowheads point to moving NtermDLK - GFP positive structures . Data information : Scale bar represents 5 μm in ( H )

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H. Representative kymograph and stills from a time lapse recording of 6DIV neurons overexpressing a GFP-tagged N-terminal region of DLK (NtermDLK-GFP). Arrowheads point to individual moving NtermDLK-GFP positive structures. Data information: Scale bar represents 5 μm in (H)


I - J . Representative Western blots of p - cJun GAPDH ( I ) , of relative c - Jun phosphorylation ( J ) from DIV6 cultured embryonic DRG neurons overexpressing GFP or NtermDLK - GFP , subjected to NGF withdrawal for 3h ( n = 7 - 8 biological replicates ; series of unpaired t / Mann - Whitney U followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

I-J. Representative Western blots of p-cJun and GAPDH (I), and quantification of relative c-Jun phosphorylation levels (J) from DIV6 cultured embryonic DRG neurons overexpressing GFP or NtermDLK-GFP, and subjected to NGF withdrawal for 3h (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


L . Representative of HeLa expressing different HA - tagged wild - type ( WT ) palmitoyl - mutant ( CS ) DLK constructs ( DLK - WT - ER , DLK - CS - ER , DLK - WT - CAAX , DLK - CS - CAAX , DLK - WT - MTS , DLK - CS - MTS ) , stained with markers of endoplasmic reticulum ( KDEL ) , plasma ( CellMask ) mitochondria ( cytochrome C ) . Data information : Scale bar represents 5 μm in ( L )

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

L. Representative images of HeLa cells expressing the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS), and stained with markers of the endoplasmic reticulum (KDEL), plasma membrane (CellMask) and mitochondria (cytochrome C). Data information: Scale bar represents 5 μm in (L)


M - N . Representative Western blots of DLK , p - cJun GAPDH ( M ) of relative c - Jun phosphorylation ( N ) from HeLa transfected with HA alone ( HA ) different HA - tagged wild - type ( WT ) palmitoyl - mutant ( CS ) DLK constructs ( DLK - WT - ER , DLK - CS - ER , DLK - WT - CAAX , DLK - CS - CAAX , DLK - WT - MTS , DLK - CS - MTS ) ( n = 7 - 8 biological replicates ; series of unpaired t / Mann - Whitney U with DLK - WT or DLK - CS followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

M-N. Representative Western blots of DLK, p-cJun and GAPDH (M) and quantification of relative c-Jun phosphorylation levels (N) from HeLa cells transfected with HA alone (HA) and the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS) (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT or DLK-CS followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


Representative Western blots of GFP , p - cJun GAPDH ( B ) from HeLa non transfected ( NT ) or transfected with GFP alone ( GFP ) , GFP - tagged DLK wild - type ( DLK - WT - GFP ) , kinase dead mutant ( DLK - KD - GFP ) , leucine zipper domain mutant ( DLK - LZ - GFP ) a palmitoyl - site mutant ( DLK - CS - GFP )

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Representative Western blots of GFP, p-cJun and GAPDH (B) from HeLa cells non transfected (NT) or transfected with GFP alone (GFP), GFP-tagged DLK wild-type (DLK-WT-GFP), kinase dead mutant (DLK-KD-GFP), leucine zipper domain mutant (DLK-LZ-GFP) and a palmitoyl-site mutant (DLK-CS-GFP)


of relative c - Jun phosphorylation ( C ) from HeLa non transfected ( NT ) or transfected with GFP alone ( GFP ) , GFP - tagged DLK wild - type ( DLK - WT - GFP ) , kinase dead mutant ( DLK - KD - GFP ) , leucine zipper domain mutant ( DLK - LZ - GFP ) a palmitoyl - site mutant ( DLK - CS - GFP ) ( n = 4 - 7 biological replicates ; series of unpaired t / Mann - Whitney U with DLK - WT - GFP followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 .

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quantification of relative c-Jun phosphorylation levels (C) from HeLa cells non transfected (NT) or transfected with GFP alone (GFP), GFP-tagged DLK wild-type (DLK-WT-GFP), kinase dead mutant (DLK-KD-GFP), leucine zipper domain mutant (DLK-LZ-GFP) and a palmitoyl-site mutant (DLK-CS-GFP) (n = 4-7 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001.


D . Representative zooms of HeLa expressing GFP alone or different GFP - tagged DLK mutants . Data information : Scale bar represents 100 μm in ( D ) zooms in ( D ) .

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D. Representative images and zooms of HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants. Data information: Scale bar represents 100 μm in (D) zooms in (D).


E - F . Representative Western blots of GFP , α - tubulin Na + / K + - ATPase from fractions ( E ) of of DLK localized in fraction ( F ) from HeLa expressing GFP alone or different GFP - tagged DLK mutants ( n = 4 - 5 biological replicates ; series of unpaired t / Mann - Whitney U with DLK - WT - GFP followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 .

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E-F. Representative Western blots of GFP, α-tubulin and Na+/K+-ATPase from cytosolic and membrane fractions (E) and quantification of the percentage of DLK localized in the membrane and cytosolic fraction (F) from HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants (n = 4-5 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001.


G - I . Representative stills ( G ) , representative kymographs ( H ) of number of GFP - positive particles ( I ) from time - lapse recordings of different GFP - tagged DLK mutants in DIV6 cultured embryonic DRG neurons ( n = 31 - 65 neurons / condition ; series of Mann - Whitney U with DLK - WT - GFP followed by a Holm - Sidak correction ) . Data information : Arrowheads point to DLK - GFP positive structures in G graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 . Scale bar represents 10 μm in ( G ) , 5 μm in ( H )

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G-I. Representative stills (G), representative kymographs (H) and quantification of the number of GFP-positive particles (I) from time-lapse recordings of the different GFP-tagged DLK mutants in DIV6 cultured embryonic DRG neurons (n = 31-65 neurons/condition; series of Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction). Data information: Arrowheads point to individual DLK-GFP positive structures in G All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001. Scale bar represents 10 μm in (G) , 5 μm in (H)


J - L . Representative stills ( J ) , kymographs ( K ) of number of DLK - GFP positive particles ( L ) from time - lapse recordings of DIV6 DRG neurons overexpressing GFP - tagged DLK treated with control ( DMSO ) or DLK inhibitor GNE - 3511 ( DLKi ) for 3h ( n = 22 - 46 neurons / condition ; Unpaired t ) . Data information : Arrowheads point to DLK - GFP positive structures in J . graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 . Scale bar represents 10 μm in ( J ) , 5 μm in ( K )

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J-L. Representative stills (J), kymographs (K) and quantification of the number of DLK-GFP positive particles (L) from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with control (DMSO) or DLK inhibitor GNE-3511 (DLKi) for 3h (n = 22-46 neurons/condition; Unpaired t test). Data information: Arrowheads point to individual DLK-GFP positive structures in J. All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001. Scale bar represents 10 μm in (J), 5 μm in (K)


A . Representative confocal deconvolved thresholded of DIV3 cultured embryonic DRG neurons co - stained for endogenous DLK ( green ) marker for synaptic vesicles Rab3 ( magenta ) . Data information : Scale bar represents , 5 μm in ( A ) 2 μm in zooms in ( A ) .

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A. Representative confocal deconvolved and thresholded images of DIV3 cultured embryonic DRG neurons co-stained for endogenous DLK (green) and the marker for synaptic vesicles Rab3 (magenta). Data information: Scale bar represents , 5 μm in (A) and 2 μm in zooms in (A).


B . Colocalization between DLK ( black values ) or same image after 90 ° rotation ( grey values ) , markers for different intracellular compartments ( Rab3 , Rab6 , Rab7 , Rab11 mitochondria ) in DIV3 cultured embryonic DRG neurons ( n = 13 - 17 / condition ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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B. Colocalization analysis between DLK (black values) or same image after 90° rotation (grey values), and markers for different intracellular compartments (Rab3, Rab6, Rab7, Rab11 and mitochondria) in DIV3 cultured embryonic DRG neurons (n = 13-17 images/condition). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


C . Representative of HeLa expressing HA alone different HA - tagged DLK constructs : a wild - type form of DLK ( DLK - WT - HA ) palmitoyl - mutant DLK ( DLK - CS - HA ) . Data information : Scale bar represents 20 μm in ( C )

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C. Representative images of HeLa cells expressing HA alone and the different HA-tagged DLK constructs: a wild-type form of DLK (DLK-WT-HA) and the palmitoyl-mutant DLK (DLK-CS-HA). Data information: Scale bar represents 20 μm in (C)


D . Western blot from total lysate ( Input ) immunoprecipitates ( IP ) of HA - tag immunoprecipitations in HeLa overexpressing HA alone different HA - tagged DLK constructs , DLK - WT - HA DLK - CS - HA in absence of any detergent .

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D. Western blot analysis from total lysate (Input) and immunoprecipitates (IP) of HA-tag protein immunoprecipitations in HeLa cells overexpressing HA alone and the different HA-tagged DLK constructs, DLK-WT-HA and DLK-CS-HA in the absence of any detergent.


E - F . Lipidomic of immunoprecipitates obtained from HA - tagged immunoprecipitations from extracts of HeLa overexpressing HA alone , DLK - WT - HA DLK - CS - HA . of lipid classes ( E ) sphingomyelin fatty acid species ( F ) measured in immunoprecipitates ( n = 3 - 4 independent experiments ; series of unpaired t / Mann - Whitney U followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . CE , cholesteryl ester ; CER , ceramide ; DAG , diacylglycerol ; DCER , dihydroceramides ; HCER , hexosylceramides , LCER , lactosylceramides ; LPC , lysophosphatidylcholine ; LPE , lysophosphatidylethanolamine ; PC , phosphatidylcholine ; PE , phosphatidylethanolamine ; SM , sphingomyelin ; TAG , triacylglycerol .

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E-F. Lipidomic analysis of the immunoprecipitates obtained from the HA-tagged protein immunoprecipitations from extracts of HeLa cells overexpressing HA alone, DLK-WT-HA and DLK-CS-HA. Quantification of lipid classes (E) and sphingomyelin fatty acid species (F) measured in the immunoprecipitates (n = 3-4 independent experiments; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. CE, cholesteryl ester; CER, ceramide; DAG, diacylglycerol; DCER, dihydroceramides; HCER, hexosylceramides, LCER, lactosylceramides; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; TAG, triacylglycerol.


G - I . Representative Western blots of DLK , p - cJun GAPDH ( G ) of relative c - Jun phosphorylation ( H ) DLK ( I ) from 3DIV cultured embryonic DRG neurons treated with D609 for 3h in presence or absence of NGF ( n = 8 biological replicates ; series of unpaired t with control NGF withdrawal condition followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

G-I. Representative Western blots of DLK, p-cJun and GAPDH (G) and quantification of relative c-Jun phosphorylation (H) and DLK levels (I) from 3DIV cultured embryonic DRG neurons treated with D609 for 3h in the presence or absence of NGF (n = 8 biological replicates; series of unpaired t tests with control NGF withdrawal condition followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


Representative stills ( J ) from time - lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP - tagged DLK treated with D609 for 3h ( n = 29 - 46 neurons Arrowheads in J point to DLK - GFP positive structures . Data information : Scale bar represents 10 μm in ( J )

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Representative stills (J) from time-lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP-tagged DLK and treated with D609 for 3h (n = 29-46 neurons Arrowheads in J point to individual DLK-GFP positive structures. Data information: Scale bar represents 10 μm in (J)


kymographs of number of DLK - GFP positive particles ( K ) from time - lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP - tagged DLK treated with D609 for 3h ( n = 29 - 46 neurons ; Mann - Whitney U ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents , 5 μm in ( K )

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kymographs and quantification of the number of DLK-GFP positive particles (K) from time-lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP-tagged DLK and treated with D609 for 3h (n = 29-46 neurons; Mann-Whitney U test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents , 5 μm in (K)


L . Representative Western blots of DLK , α - tubulin Na + / K + - ATPase from , insoluble fractions of 3DIV cultured embryonic DRG neurons treated with D609 for 3h .

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

L. Representative Western blots of DLK, α-tubulin and Na+/K+-ATPase from cytosolic, membrane and insoluble fractions of 3DIV cultured embryonic DRG neurons treated with D609 for 3h.


A . Western blot of endogenous DLK , p - cJun , Rab6 GAPDH , of relative c - Jun phosphorylation in 3DIV cultured embryonic DRG neurons treated with dynasore ( DYN ) or brefeldin A ( BFA ) for 3h in presence or absence of NGF ( n = 6 - 12 biological replicates ; series of unpaired t / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

A. Western blot analysis of endogenous DLK, p-cJun, Rab6 and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) or brefeldin A (BFA) for 3h in the presence or absence of NGF (n = 6-12 biological replicates; series of unpaired t tests /Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


B . Representative Western blots of p - MKK4 , MKK4 GAPDH , quantifications of relative MKK4 phosphorylation in 3DIV cultured embryonic DRG neurons treated with dynasore ( DYN ) for 3h in presence or absence of NGF ( n = 6 biological replicates , series of unpaired t with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

B. Representative Western blots of p-MKK4, MKK4 and GAPDH, and quantifications of the relative MKK4 phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) for 3h in the presence or absence of NGF (n = 6 biological replicates, series of unpaired t tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


Representative stills ( C ) , kymographs ( D ) of orientation ( E ) of DLK - GFP positive particles from time - lapse recordings of DIV6 DRG neurons overexpressing GFP - tagged DLK treated with dynasore ( DYN ) or brefeldin A ( BFA ) for 3h ( n = 34 - 101 neurons , Chi - square in E Arrowheads in C point to DLK - GFP positive structures . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 10 μm in ( C ) , 5 μm in ( D )

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

Representative stills (C), kymographs (D) and quantification of the orientation (E) of DLK-GFP positive particles from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with dynasore (DYN) or brefeldin A (BFA) for 3h (n = 34-101 neurons, Chi-square test in E Arrowheads in C point to individual DLK-GFP positive structures. Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (C) and , 5 μm in (D)


of number of DLK - GFP positive particles ( F ) from time - lapse recordings of DIV6 DRG neurons overexpressing GFP - tagged DLK treated with dynasore ( DYN ) or brefeldin A ( BFA ) for 3h ( n = 34 - 101 neurons , series of Mann - Whitney with control followed by a Holm - Sidak correction in F ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

quantification of number of DLK-GFP positive particles (F) from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with dynasore (DYN) or brefeldin A (BFA) for 3h (n = 34-101 neurons, series of Mann-Whitney tests with control followed by a Holm-Sidak correction in F). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


G - H . Representative Western blots of DLK , α - tubulin Na + / K + - ATPase ( G ) quantifications ( H ) from fractions of 3DIV cultured embryonic DRG neurons with dynasore ( DYN ) for 3h in absence of NGF ( n = 5 - 11 biological replicates , series of unpaired t / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

G-H. Representative Western blots of DLK, α-tubulin and Na+/K+-ATPase (G) and quantifications (H) from cytosolic and membrane fractions of 3DIV cultured embryonic DRG neurons with dynasore (DYN) for 3h in the absence of NGF (n = 5-11 biological replicates, series of unpaired t tests /Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


I - K . Representative stills ( I ) , kymographs ( J ) of number of DLK - GFP positive particles ( K ) from cultured embryonic DRG neurons treated with chlorpromazine ( CHP ) , dansylcadaverine ( DCV ) or methyl - β - cyclodextrin ( MCD ) for 3h ( n = 12 neurons , series of Mann - Whitney U with control situation followed by a Holm - Sidak correction ) . Arrowheads in I point to DLK - GFP positive structures . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 10 μm in ( I ) , 5 μm in ( J ) .

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

I-K. Representative stills (I), kymographs (J) and quantification of the number of DLK-GFP positive particles (K) from cultured embryonic DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 3h (n = 12 neurons, series of Mann-Whitney U tests with control situation followed by a Holm-Sidak correction). Arrowheads in I point to individual DLK-GFP positive structures. Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (I), 5 μm in (J).


L . Representative Western blots of p - cJun GAPDH , of relative c - Jun phosphorylation in 3DIV cultured embryonic DRG neurons treated with chlorpromazine ( CHP ) , dansylcadaverine ( DCV ) or methyl - β - cyclodextrin ( MCD ) for 3h in absence of NGF ( n = 6 - 24 biological replicates , series of Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

L. Representative Western blots of p-cJun and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 3h in the absence of NGF (n = 6-24 biological replicates, series of Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


M . Representative Western blots of p - cJun GAPDH , of relative c - Jun phosphorylation in 3DIV cultured embryonic DRG neurons treated with vincristine ( VNC ) dynasore ( DYN ) for 3h ( n = 7 biological replicates , series of Mann - Whitney U with vincristine condition followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

M. Representative Western blots of p-cJun and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with vincristine (VNC) and dynasore (DYN) for 3h (n = 7 biological replicates, series of Mann-Whitney U tests with vincristine condition followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


A . Western blot of caspase - 3 ( cCasp3 ) GAPDH , quantifications of cCasp3 in 3DIV cultured embryonic DRG neurons treated with dynasore ( DYN ) for 8h in absence of NGF ( n = 6 - 13 biological replicates , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

A. Western blot analysis of cleaved caspase-3 (cCasp3) and GAPDH, and quantifications of cCasp3 levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) for 8h in the absence of NGF (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


B . Quantifications of cCasp3 from 3DIV DRG neurons treated with chlorpromazine ( CHP ) , dansylcadaverine ( DCV ) or methyl - β - cyclodextrin ( MCD ) for 8h in absence of NGF ( n = 6 - 13 biological replicates , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

B. Quantifications of cCasp3 levels from 3DIV DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 8h in absence of NGF (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


C . Representative of DRG neurons expressing GFP , Dnm - 1 - WT - HA or Dnm1 - DN - HA form , cultured in absence of NGF for 8h , stained with antibodies against cCasp3 ( magenta ) HA tag ( green ) . On right , of of cCasp3 positive over GFP or HA positive ( n = 10 - 15 from 3 independent cultures , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 20 μm in ( C )

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

C. Representative images of DRG neurons expressing GFP, Dnm-1-WT-HA or Dnm1-DN-HA form, cultured in the absence of NGF for 8h, and stained with antibodies against cCasp3 (magenta) and HA tag (green). On the right, quantification of the ratio of cCasp3 positive cells over GFP or HA positive cells (n = 10-15 images from 3 independent cultures, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 20 μm in (C)


D . Representative of axons from E12 . 5 DRG explants cultured for 2 days , subsequently deprived of NGF treated with different concentrations of dynasore ( DYN ) for 24 h , visualized by immunostaining for βIII - tubulin . of axonal degeneration index as of fragmented axon over total axon ( n = 13 - 15 explants , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 20 μm in ( D ) 10 μm in zooms in ( D ) .

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

D. Representative images of axons from E12.5 DRG explants cultured for 2 days, subsequently deprived of NGF and treated with different concentrations of dynasore (DYN) for 24 h, and visualized by immunostaining for βIII-tubulin. Quantification of the axonal degeneration index as the ratio of fragmented axon area over total axon area (n = 13-15 explants, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 20 μm in (D) and 10 μm in zooms in (D).


E . Representative of axons from cultured embryonic DRG neurons cultured for 2 days , subsequently deprived of NGF treated with different concentrations of dynasore ( DYN ) for 24 h , visualized by imaging SiR - tubulin probe . On right , of fragmented axons using degeneration index ( n = 15 from 3 independent cultures , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 100 μm in ( E )

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 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 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

E. Representative images of axons from cultured embryonic DRG neurons cultured for 2 days, subsequently deprived of NGF and treated with different concentrations of dynasore (DYN) for 24 h, and visualized by imaging SiR-tubulin probe. On the right, quantification of fragmented axons using the degeneration index (n = 15 images from 3 independent cultures, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 100 μm in (E)


F . Representative of axons from cultured embryonic DRG neurons expressing GFP , Dnm - 1 - WT - HA or Dnm1 - DN - HA form deprived of NGF for 24h , visualized with an antibody against neurofilament heavy chain ( NFH ) . On right , of fragmented axons using degeneration index in ( n = 8 - 13 from 3 independent cultures , series of unpaired t - with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 40 μm in ( F )

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

F. Representative images of axons from cultured embryonic DRG neurons expressing GFP, Dnm-1-WT-HA or Dnm1-DN-HA form and deprived of NGF for 24h, and visualized with an antibody against neurofilament heavy chain (NFH). On the right, quantification of fragmented axons using the degeneration index in (n = 8-13 images from 3 independent cultures, series of unpaired t-test tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 40 μm in (F)


A , HBMEC were challenged with type I ( RH ) or type II ( ME49 ) strains of T . gondii for indicated time points . lysates were used to probe for total Akt phospho - Ser473 Akt by immunoblot .

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A, Primary HBMEC were challenged with type I (RH) or type II (ME49) strains of T. gondii for the indicated time points. Cell lysates were used to probe for total Akt and phospho-Ser473 Akt by immunoblot.


B , C , Mouse endothelial ( mHEVc ; B ) human retinal pigment epithelial ( RPE , C ) were challenged with RH T . gondii followed by assessment of Akt phosphorylation .

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B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation.


D , HBMEC were challenged with either live or killed T . gondii . lysates were subject to immunoblotting as indicated .

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D, HBMEC were challenged with either live or killed T. gondii. Cell lysates were subject to immunoblotting as indicated.


E , HBMEC were transiently transfected with a plasmid encoding PH - Akt - GFP . treated with or without LY294002 ( 20 µM ) for 1 h prior to challenge with T . gondii - RFP . were examined by immunofluorescence at 5 min post - challenge to examine accumulation of PH - Akt - GFP around .

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E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites.


F , HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T . gondii . Densitometry data represent means ± of 4 experiments . A vertical was inserted between densitometry data from control LY294002 - treated to indicate that band densities from infected treated with or without LY294002 are compared to bands from their respective uninfected , which were given an arbitrary number of 1 .

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F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1.


G , HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA . expressing either control siRNA or PI3K p110α siRNA were challenged with T . gondii 48 h after transfection . lysates were subject to immunoblotting as indicated . Densitometry data are shown as above represent means ± of 3 experiments . Results shown are representative of 3 - 4 independent experiments .

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G, HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA. Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T. gondii 48 h after transfection. Cell lysates were subject to immunoblotting as indicated. Densitometry data are shown as above and represent means ± SEM of 3 experiments. Results shown are representative of 3-4 independent experiments.


A , HBMEC , mHEVc human RPE were incubated with or without Akt inhibitor IV ( 1 . 25 µM ) for 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy 2 h 24 h post - challenge .

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A, HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


B , HBMEC were transfected with control siRNA or Akt siRNA . were then challenged with T . gondii 48 h after transfection . Monolayers were examined microscopically 24 h post - challenge .

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B, HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge.


C , RAW 264 . 7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy 2 h 24 h post - challenge .

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C, RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


D , mHEVc - LC3 - EGFP were incubated with or without Akt inhibitor IVfollowed by challenge with T . gondii - RFP . Monolayers were examined by microscopy 5 h post - challenge . Arrowheads indicate accumulation of LC3 around .

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D, mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite.


E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii ( T ) then processed for electron microscopy at 5 h post - challenge . at bottom represent magnification of areas within boxes . Arrow indicates PVM ; arrowhead indicates double structure around vacuole .

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E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole.


F , Control or Akt inhibitor IV - treated HBMEC were challenged with T . gondii - YFP . Expression of LAMP - 1 was examined by microscopy 8 h post - challenge . Arrowheads indicate accumulation of LAMP - 1 around

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F, Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite


G , H , mHEVc were transfected with Beclin 1 siRNA ( G ) , Atg7 siRNA ( H ) or control siRNA . After 48 h , were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy at 24 h .

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G, H, mHEVc cells were transfected with Beclin 1 siRNA (G), Atg7 siRNA (H) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 24 h.


I , mHEVc were treated with or without Akt inhibitor IV infected with T . gondii . 1 h post infection were treated with or without leupeptin plus pepstatin ( Lys inhibitors ) . Monolayers were examined microscopically 24 h post - challenge .

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I, mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii. 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge.


J , Mouse microglia were incubated with or without Akt inhibitor IV . 3 - methyl adenine ( 3MA ; 10 mM ) or vehicle were added 2 h post - challenge with T . gondii . Monolayers were examined microscopically 24 h post - challenge . Results are shown as mean ± are representative of 3 independent experiments .

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J, Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii. Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


A , HBMEC were treated with or without pertussis toxin ( PTx ; 100 ng / mL ) for 4 h prior to challenge with T . gondii . lysates were obtained at 5 , 15 or 30 min post - incubation with T . gondii used to examine total Akt phospho - Akt serine 473 by immunoblot . Densitometry data represent means ± of 3 experiments . A vertical was inserted between densitometry data from control PTx - treated to indicate that band densities from infected treated with or without PTx were compared to bands from their respective uninfected , which were given an arbitrary number of 1 . HBMEC were also treated with or without LPA ( 10 µM ) in presence or absence of PTx . lysates were obtained at 5 min subjected to

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A, HBMEC were treated with or without pertussis toxin (PTx; 100 ng/mL) for 4 h prior to challenge with T. gondii. Cell lysates were obtained at 5, 15 or 30 min post-incubation with T. gondii and used to examine total Akt and phospho-Akt serine 473 by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and PTx-treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. HBMEC were also treated with or without LPA (10 µM) in the presence or absence of PTx. Cell lysates were obtained at 5 min and subjected to immunoblotting


B , HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h . lysates were used to examine total Akt phospho - serine 473 Akt by immunoblot . Results shown are representative of 3 independent experiments .

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B, HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h. Cell lysates were used to examine total Akt and phospho-serine 473 Akt by immunoblot. Results shown are representative of 3 independent experiments.


A , HBMEC were challenged with RH T . gondii lysates were obtained to probe for total EGFR , phospho - tyrosine 1068 EGFR phospho - tyrosine 1148 EGFR by immunoblot .

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A, HBMEC cells were challenged with RH T. gondii and cell lysates were obtained to probe for total EGFR, phospho-tyrosine 1068 EGFR and phospho- tyrosine 1148 EGFR by immunoblot.


B , HBMEC were challenged with live vs killed tachyzoites . Total EGFR phospho - tyrosine 1068 EGFR expression was examined by immunoblot .

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B, HBMEC were challenged with live vs killed tachyzoites. Total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot.


C , D , Human RPE ( C ) mouse microglia ( D ) were challenged with RH T . gondii total EGFR phospho - tyrosine 1068 EGFR expression was examined by immunoblot . Results shown are representative of 3 independent experiments .

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C, D, Human RPE cells (C) and mouse microglia (D) were challenged with RH T. gondii and total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot. Results shown are representative of 3 independent experiments.


A , mHEVc were transfected with control siRNA or EGFR siRNA . Expression of EGFR actin were assessed by immunoblot 48 h post - transfection . mHEVc expressing either control siRNA or EGFR siRNA were challenged with T . gondii . lysates were used to examine expression total Akt or phospho - Ser473 Akt by immunoblot .

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A, mHEVc cells were transfected with control siRNA or EGFR siRNA. Expression of EGFR and actin were assessed by immunoblot 48 h post-transfection. mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot.


B , Mouse microglia were treated with or without AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . lysates were used to examine expression total Akt or phospho - Ser473 Akt by immunoblot . Densitometry data represent means ± of 3 experiments . A vertical was inserted between densitometry data from control siRNA EGFR siRNA or control AG1478 - treated to indicate that band densities from infected control or infected subjected to EGFR blockade were compared to bands from their respective uninfected , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


A , HBMEC were incubated with AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy at 2 24 h .

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A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


B , Human RPE transfected with either EGFR or control siRNA were challenged with T . gondii followed by examination of monolayers by microscopy at 24 h .

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B, Human RPE cells transfected with either EGFR or control siRNA were challenged with T. gondii followed by examination of monolayers by light microscopy at 24 h.


C , Mouse bone marrow - derived macrophages were incubated with AG1478 challenged with T . gondii . Monolayers were examined by microscopy at 2 24 h .

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C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


D , Parental CHO CHO - EGFR were challenged with RH T . gondii . Monolayers were examined microscopically 2 h or 24 h post - challenge .

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D, Parental CHO and CHO-EGFR cells were challenged with RH T. gondii. Monolayers were examined microscopically 2 h or 24 h post-challenge.


E , mHEVc - LC3 - EGFP transfected with control siRNA or EGFR siRNA were challenged with T . gondii - RFP . Monolayers were examined by microscopy 5 h post - challenge to determine of endothelial with LC3 accumulation around .

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E, mHEVc-LC3-EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite.


F , HBMEC treated with or without AG1478 were challenged with either T . gondii - YFP . Expression of LAMP - 1 was examined by microscopy 8 h post - challenge . percentages of endothelial with LAMP - 1 accumulation of around were determined .

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F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined.


G , H , mHEVc transfected with Beclin1 siRNA ( G ) or Atg7 siRNA ( H ) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T . gondii . Monolayers were examined by microscopy 24 h post - challenge . Results are shown as mean ± are representative of 3 independent experiments .

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G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


HBMEC were treated with or without GM6001 ( 10 µM ) for 1 h prior to challenge with T . gondii ( A ) or with Pertussis Toxin ( PTx ; 100 ng / ml ) for 4 h prior to challenge ( B ) . lysates were used to examine total EGFR phospho - tyrosine 1068 EGFR by immunoblot . Densitometry data represent means ± of 3 experiments . A vertical was inserted between densitometry data from control GM6001 - or PTX - treated to indicate that band densities from infected treated with or without these inhibitors are compared to bands from their respective uninfected , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


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A , B , C HBMEC were challenged with ΔHx ( WT ) , MIC1 ko , MIC3 ko , MIC1 - 3 ko T . gondii at MOIs that yielded similar percentages of infected ( A ) .

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A, B, C HBMEC were challenged with ΔHx (WT), MIC1 ko, MIC3 ko, MIC1-3 ko T. gondii at MOIs that yielded similar percentages of infected cells (A).


lysates were obtained used to examine total EGFR phospho - tyrosine 1068 EGFR ( B ) or total Akt phospho - Ser 473 Akt ( C ) by immunoblot .

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Cell lysates were obtained and used to examine total EGFR and phospho-tyrosine 1068 EGFR (B) or total Akt and phospho-Ser 473 Akt (C) by immunoblot.


HBMEC were incubated with 10 nM of recombinant MIC4 , MIC3 , M2AP or MIC6 for 15 minutes followed by examination of total EGFR phospho - tyrosine 1068 EGFR ( D ) or total Akt phospho - Ser473 ( E ) by immunoblot . Densitometry data represent means ± of 4 experiments . Band densities from infected or treated with MICs were compared to bands from uninfected or untreated ( Un ) , which were given an arbitrary number of 1 . Results shown are representative of 4 independent experiments .

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HBMEC were incubated with 10 nM of recombinant MIC4, MIC3, M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho-tyrosine 1068 EGFR (D) or total Akt and phospho-Ser473 (E) by immunoblot. Densitometry data represent means ± SEM of 4 experiments. Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells (Un), which were given an arbitrary number of 1. Results shown are representative of 4 independent experiments.


A , hmCD40 mHEVc expressing LC3 - EGFP were treated with or without CD154 followed by challenge with T . gondii - RFP in presence or absence of EGF ( 50 ng / ml ) or AG1478 ( 1 µM ) . LC3 accumulation around T . gondii was assessed by immunofluorescence . B , mHEVc - LC3 - EGFP were infected with T . gondii - T . gondii . were treated with vehicle or rapamycin ( 1 µM ) 2 h after challenge with T . gondii in presence or absent of EGF or AG1478 . Recruitment of LC3 around was examined at 5 h after challenge with T . gondii .

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A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-T. gondii. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii.


. C , hmCD40 mHEVc - LC3 - EGFP were treated with or without CD154 followed by challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko , MIC1 - 3 ko + MIC1 - 3 tachyzoites . LC3 accumulation around was examined by microscopy 5 h post - challenge . D , hmCD40 mHEVc - LC3 - EGFP treated with or without CD154 were challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko or MIC1 - 3 ko + MIC1 - 3 tachyzoites . load was examined 24 h post - challenge .

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. C, hmCD40 mHEVc-LC3-EGFP cells were treated with or without CD154 followed by challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko, MIC1-3 ko+MIC1-3 tachyzoites. LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post-challenge. D, hmCD40 mHEVc-LC3-EGFP treated with or without CD154 were challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko or MIC1-3 ko+MIC1-3 tachyzoites. Parasite load was examined 24 h post-challenge.


E , HBMEC were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites followed by treatment with vehicle or rapamycin . load was examined 24 h post - challenge . F , HBMEC treated with or without CD154 or IFN - γ / TNF - α were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites . Monolayers were examined microscopically at 24 h post - challenge .

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E, HBMEC cells were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites followed by treatment with vehicle or rapamycin. Parasite load was examined 24 h post-challenge. F, HBMEC treated with or without CD154 or IFN-γ/TNF-α were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites. Monolayers were examined microscopically at 24 h post-challenge.


G , hmCD40 mHEVc treated with or without CD154 were infected with either WT or MIC1 - 3 ko tachyzoites in presence of complete medium ( CM ) alone or medium plus MIC3 , MIC4 , M2AP or MIC6 ( 10 nM ) . load was determined microscopically 24 h post challenge . Results shown are representative of 3 - 4 independent experiments .

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G, hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1-3 ko tachyzoites in the presence of complete medium (CM) alone or medium plus MIC3, MIC4, M2AP or MIC6 (all 10 nM). Parasite load was determined microscopically 24 h post challenge. Results shown are representative of 3-4 independent experiments.


exclusion chromatography profiles of recombinant used in this study . AU : arbitrary units ; WT : wild type ; FL : full - .

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Size exclusion chromatography profiles of recombinant proteins used in this study. AU: arbitrary units; WT: wild type; FL: full-length.


of interaction between CtUba4 GST - CtUrm1 ( 2 : 1 or 0 . 5 : 1 when marked with an asterisk ) in presence of nucleotide derivates by GST pull - down resolved by SDS - PAGE visualized with Coomassie stain .

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Analysis of the interaction between CtUba4 and GST-CtUrm1 (ratio 2:1 or 0.5:1 when marked with an asterisk) in the presence of nucleotide derivates by GST pull-down resolved by SDS-PAGE and visualized with Coomassie stain.


Functional analyses of ScUba4 mutant yeast using APM - gel retardation ( top ) viability in response to rapamycin ( bottom ) . PAGE was supplemented with APM to retard migration of thiolated tRNAs allow their visualization by northern blot using an anti - tRNA < sub > UU < em > C < / em > < / sub > < sup > Gl < em > u < / em > < / sup > ; probe . APM : ( [ N - Acryloyl - ] phenyl ) mercuric chloride ; YPD : yeast extract peptone dextrose . residue numbering follows CtUba4 , but respective ScUba4 numbering is added in subscript .

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Functional analyses of ScUba4 mutant yeast using APM-gel retardation (top) and viability in response to rapamycin (bottom). PAGE was supplemented with APM to retard the migration of thiolated tRNAs and allow their visualization by northern blot using an anti-tRNAUUCGlu; probe. APM: ([N-Acryloyl-amino]phenyl)mercuric chloride; YPD: yeast extract peptone dextrose. All residue numbering follows the CtUba4 sequence, but the respective ScUba4 numbering is added in subscript.


Interaction of WT mutated CtUba4 with GST - CtUrm1 by GST pull - down in presence of 1mM ATP . CtUba4C202K covalently linked to GST - CtUrm1 is marked by an asterisk .

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Interaction analysis of WT and mutated CtUba4 with GST-CtUrm1 by GST pull-down in the presence of 1mM ATP. CtUba4C202K covalently linked to GST-CtUrm1 is marked by an asterisk.


of covalent adduct formation between CtUba4 WT or C202 mutants carboxylated ( - OH ) or thiocarboxylated ( - SH ) CtUrm1 in presence or absence of TBH , DTT , TCEP HA , respectively . ATP : Adenosine triphosphate TBH : tert - Butyl hydroperoxide HA : Hydroxylamine ; DTT : 1 , 4 - Dithiothreitol ; TCEP : Tris ( 2 - carboxyethyl ) phosphine .

Analysis of covalent adduct formation between CtUba4 WT or C202 mutants and carboxylated (-OH) or thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH, DTT, TCEP and HA, respectively. ATP: Adenosine triphosphate TBH: tert-Butyl hydroperoxide HA: Hydroxylamine; DTT: 1,4-Dithiothreitol; TCEP: Tris(2-carboxyethyl)phosphine.


of covalent adduct formation between CtUba4 WT / cysteine mutants CtUrm1 - COSH in presence of oxidizing agents . HP : Hydrogen peroxide ; D : Diamide .

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Analysis of covalent adduct formation between CtUba4 WT/cysteine mutants and CtUrm1-COSH in the presence of oxidizing agents. HP: Hydrogen peroxide; D: Diamide.


Phenotypic of ScUba4 mutant yeast strains in response to rapamycin .

Phenotypic analysis of ScUba4 mutant yeast strains in response to rapamycin.


MS / MS spectrum of K202 - ε - GGH containing peptide from CtUba4C202K . A representative annotated fragmentation spectrum is shown with b - y - ions marked in red blue , respectively . precursor ion at m / z 591 , 98 is labelled in green . peptide is shown at top with collision - induced fragmentation pattern . Urm1 conjugation site was identified by detection of GGH remnant motif generated by chymotryptic digestion .

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

MS/MS spectrum of the K202-ε-GGH containing peptide from CtUba4C202K. A representative annotated fragmentation spectrum is shown with the b- and y-ions marked in red and blue, respectively. The precursor ion at m/z 591,98 is labelled in green. The peptide sequence is shown at the top with the collision-induced fragmentation pattern. The Urm1 conjugation site was identified by detection of the GGH remnant motif generated by chymotryptic digestion.


of covalent adduct formation between CtUba4 WT or Cys202 mutants thiocarboxylated ( - SH ) CtUrm1 in presence or absence of TBH HA , respectively .

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Analysis of covalent adduct formation between CtUba4 WT or Cys202 mutants and thiocarboxylated (-SH) CtUrm1 in the presence or absence of TBH and HA, respectively.


( A ) Genome - wide ubiquitin binding profiles identify numerous regions of H2B - ubiquitylation in WT distinct sites of non - H2B ubiquitylation ( ' ubiquitin - hotspot ' , ub - hotspot , ub - HS ) which persist in h2b - K123R rad6 ∆ strains , increase in cdc48 mutants ( cdc48 - 6 ) . A 90 kb stretch of chromosome XIII ( ChrXIII ) is depicted . immunoprecipitation was performed using FK2 ubiquitin antibody ( see also Appendix Fig . S1A ) enriched was analyzed on NimbleGen arrays ( Chip - chip ) . from non - specific IgG - ChIP experiments served as background control . Significantly enriched regions are marked by bars above respective ChIP - chip tracks are summarized in Dataset EV1 . Data represent means from two independent replicates , except for ubiquitin ( FK2 ) in rad6 ∆ IgG - ChIP in WT ( n = 1 ) . experiments , including those using cdc48 - 6 other - sensitive ( ts ) alleles , were performed at 30 ° C ( semipermissive for ts - alleles ) unless stated otherwise .

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

(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise.


( B ) Seven ub - HSs show strong correlation between ubiquitin binding Slx8 enrichment ( Slx8 - 9myc ChIP ) . 16 - kb - windows of indicated regions centered around ub - HSs are depicted for ubiquitin Slx8 binding profiles . Ubiquitin ( FK2 ) data for WT cdc48 - 6 are from same experiment as depicted in ( A ) . Data represent means from two independent replicates .

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(B) Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb-windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates.


( D ) Slx8 Slx5 are recruited to ub - HSs . from Slx8 - 9myc ( top ) or 3HA - Slx5 ( bottom ) ChIP experiments of indicated strains was analyzed by quantitative real - time PCR ( ChIP - qPCR ) at selected ub - HSs . Data represent mean ± standard deviation ( SD , n = 2 ) . Consistent with a previous study , we did not observe any Slx8 enrichment at centromeres ( van de Pasch et al , 2013 ) , however , we note that we could not confirm reported centromere binding of Slx5 ( see also Appendix Fig . S2E ) .

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

(D) Slx8 and Slx5 are recruited to ub-HSs. DNA from Slx8-9myc (top) or 3HA-Slx5 (bottom) ChIP experiments of the indicated strains was analyzed by quantitative real-time PCR (ChIP-qPCR) at selected ub-HSs. Data represent mean ± standard deviation (SD, n = 2). Consistent with a previous study, we did not observe any Slx8 enrichment at centromeres (van de Pasch et al, 2013), however, we note that we could not confirm the reported centromere binding of Slx5 (see also Appendix Fig. S2E).


( E ) K48 - linked ubiquitin chains accumulate in a cdc48 mutant ( cdc48 - 3 ) . Ub - K48 ChIP followed by qPCR for same loci as in ( D ) are depicted . See also Fig . EV1C - D . Data represent means ± SD ( n = 2 ) .

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(E) K48-linked ubiquitin chains accumulate in a cdc48 mutant (cdc48-3). Ub-K48 ChIP followed by qPCR for same loci as in (D) are depicted. See also Fig. EV1C-D. Data represent means ± SD (n = 2).


( F ) Slx5 Slx8 are required for ub - HS formation . Ub - K48 ChIP - qPCR in strains lacking one subunit of Slx5 / Slx8 ( slx5 ∆ , slx8 ∆ ) . Data represent means ± SD ( n = 3 ) .

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(F) Slx5 and Slx8 are required for ub-HS formation. Ub-K48 ChIP-qPCR in strains lacking one subunit of the Slx5/Slx8 complex (slx5∆, slx8∆). Data represent means ± SD (n = 3).


( G ) Ufd1 Npl4 act in concert with Cdc48 to remove ubiquitylated from ub - HS sites . Ub - K48 ChIP for indicated strains grown at semi - permissive of 30 ° C . Data represent means ± SD ( n = 2 ) .

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(G) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub-HS sites. Ub-K48 ChIP for the indicated strains grown at the semi-permissive temperature of 30°C. Data represent means ± SD (n = 2).


B ) A 39 bp fragment of ub - HS4 is sufficient to drive ectopic ub - HS formation . Fragments of ub - HS4 were integrated ectopically ub - K48 ChIP - qPCR was performed for endogenous ub - HS5 ectopic ub - HS4 fragments as depicted in ( A ) . contr . : control , empty YI128 - vector was integrated at LEU2 . Experiments were performed in cdc48 ‑ 6 background . Data represent means ± SD ( n = 2 ) . Experimental mapping bioinformatic prediction identify a similar ub - HS - motif . Comparison between experimentally mapped ub - HS4 F7 ( B ) consensus motif of ub - HSs identified by MEME software .

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B) A 39 bp fragment of ub-HS4 is sufficient to drive ectopic ub-HS formation. Fragments of ub-HS4 were integrated ectopically and ub-K48 ChIP-qPCR was performed for the endogenous ub-HS5 and the ectopic ub-HS4 fragments as depicted in (A). contr.: control, empty YI128-vector was integrated at LEU2. Experiments were performed in cdc48‑6 background. Data represent means ± SD (n = 2). Experimental mapping and bioinformatic prediction identify a similar ub-HS-motif. Comparison between the experimentally mapped ub-HS4 F7 (B) and the consensus motif of all ub-HSs identified by the MEME software.


E ) Euc1 binds to ub - HS - motif in a Y1H . Gal4 - AD - or Gal4 - AD - Euc1 - encoding plasmids were transformed into a Y1H reporter as described in ( D ) . Serial dilutions were spotted on control plates plates lacking histidine with 20mM 3 - - triazole ( 3AT ) . were grown at 30 ° C for 3 days .

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E) Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days.


F ) Euc1 is required for formation of ub - HSs . ChIP with ub - K48 specific antibodies was performed for indicated strains enriched was analyzed by qPCR . Data represent means ± SD ( n = 4 ) .

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F) Euc1 is required for formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4).


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of K48 - linked ubiquitin chains were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of K48-linked ubiquitin chains were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of Euc1 were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of Euc1 were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


( C ) Euc1 can induce transactivation via its N - terminal 30 . Euc1 constructs under endogenous EUC1 promoter were transformed in a reporter as described for Fig . 2D serial dilutions were spotted on control or selective media to HIS3 - activation . were grown at 30 ° C for 3 days .

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(C) Euc1 can induce transactivation via its N-terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig. 2D and serial dilutions were spotted on control or selective media to test HIS3-activation. Cells were grown at 30°C for 3 days.


( D ) of HIS3 mRNA from strains used in ( C ) . were grown in liquid media with selection for transformed plasmids ( SC - Leu ) , harvested in logarithmic growth phase total mRNA was prepared . After reverse transcription , HIS3 mRNA were quantified using qPCR ( RT - qPCR ) , normalized first to ACT1 mRNA then to empty vector control . Data represent means ± SD ( n = 4 ) . p = 2 . 43 * 10 - 5 ( Student ' s t - ) .

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(D) Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC-Leu), harvested in logarithmic growth phase and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT-qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). p = 2.43*10-5 (Student's t-test).


( A ) Euc1 interacts with SUMO pathway in a yeast two - hybrid ( Y2H ) . A Y2H reporter ( PJ69 - 7a ) was transformed with Gal4 - binding domain ( BD ) Gal4 activation domain ( AD ) fusion constructs in indicated combinations . AD - SUMO - GG can be conjugated to SUMOylation substrates , while AD - SUMO - AA is conjugation - deficient . were spotted on control media or selective media ( - His ) grown for 3 6 days , respectively .

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(A) Euc1 interacts with SUMO pathway proteins in a yeast two-hybrid (Y2H) assay. A Y2H reporter strain (PJ69-7a) was transformed with Gal4 DNA-binding domain (BD) and Gal4 activation domain (AD) fusion constructs in the indicated combinations. AD-SUMO-GG can be conjugated to SUMOylation substrates, while AD-SUMO-AA is conjugation-deficient. Cells were spotted on control media or selective media (- His) and grown for 3 and 6 days, respectively.


( B ) Euc1 is SUMOylated by Ubc9 , Siz1 Siz2 on K231 . Euc1 was immunoprecipitated from indicated strains eluates were probed by WB against SUMO ( top ) Euc1 ( bottom ) . An up - shifted band corresponding to Euc1SUMO was detected with both antibodies . Asterisks denote non - specific bands .

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(B) Euc1 is SUMOylated by Ubc9, Siz1 and Siz2 on K231. Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO (top) and Euc1 (bottom). An up-shifted band corresponding to Euc1SUMO was detected with both antibodies. Asterisks denote non-specific bands.


( C ) Euc1 is predominantly monoSUMOylated on lysine 231 . Denaturing NiNTA - pulldowns ( NiNTA - PD ) with strains expressing His - tagged SUMO ( HisSUMO ) as indicated 3FLAGEuc1 constructs under control of an ADH promoter . Covalently SUMO - modified were enriched eluates probed with a FLAG antibody to visualize SUMOylated Euc1 . Eluates were probed for SUMO to control for equal pull - down , Pgk1 was probed as input control . Euc1 - KR denotes K231R mutation here hereafter .

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(C) Euc1 is predominantly monoSUMOylated on lysine 231. Denaturing NiNTA-pulldowns (NiNTA-PD) with strains expressing His-tagged SUMO (HisSUMO) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter. Covalently SUMO-modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1. Eluates were probed for SUMO to control for equal pull-down, Pgk1 was probed as input control. Euc1-KR denotes the K231R mutation here and hereafter.


( D ) Euc1 SUMOylation is required for ub - HS formation . Ub - K48 ChIP quantified by qPCR for indicated strains . ubx5 ∆ background was used to enhance ubiquitin signal , similar results were obtained in a WT background . Data represent means ± SD ( n = 3 ) .

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(D) Euc1 SUMOylation is required for ub-HS formation. Ub-K48 ChIP quantified by qPCR for the indicated strains. The ubx5∆ background was used to enhance the ubiquitin signal, similar results were obtained in a WT background. Data represent means ± SD (n = 3).


( E ) Euc1 ubiquitylation depends on Slx8 partly on Euc1 SUMOylation . Denaturing NiNTA - PDs as described for ( C ) but with strains expressing His - ubiquitin ( HisUbi ) 3FLAGEuc1 under control of ADH promoter . WBs were developed with a FLAG antibody to probe for 3FLAGEuc1 , a ubiquitin blot served as PD - control Dpm1 as input control . slx8 - CD allele carries C206S , C209S - mutations ( Xie et al , 2007 ) .

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(E) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation. Denaturing NiNTA-PDs as described for (C) but with strains expressing His-ubiquitin (HisUbi) and 3FLAGEuc1 under the control of the ADH promoter. WBs were developed with a FLAG antibody to probe for 3FLAGEuc1, a ubiquitin blot served as PD-control and Dpm1 as input control. The slx8-CD allele carries the C206S, C209S-mutations (Xie et al, 2007).


SUMOylated Euc1 is required to recruit Slx5 Slx8 to ub - HSs . ChIP against Slx5 in indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT 3HA - Slx5 in ( F )

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SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub-HSs. ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and 3HA-Slx5 in (F) and WT


SUMOylated Euc1 is required to recruit Slx8 to ub - HSs . ChIP against Slx8 in indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT Slx8 - 9myc in ( G )

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SUMOylated Euc1 is required to recruit Slx8 to ub-HSs. ChIP against Slx8 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and Slx8-9myc in (G)


( A ) Euc181 - 183 binds to Slx5 in Y2H assays . Y2H performed as in Fig . 4A . Serial dilutions were spotted for each plasmid combination were grown at 30 ° C for 2 days . Slx5 - RING ∆ construct is deleted for Slx5 C - terminus from beginning of RING - domain ( ∆ aa 488 - 619 ) . See also Fig . EV3B .

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(A) Euc181-183 binds to Slx5 in Y2H assays. Y2H assay performed as in Fig. 4A. Serial dilutions were spotted for each plasmid combination and cells were grown at 30°C for 2 days. The Slx5-RING∆ construct is deleted for the Slx5 C-terminus from beginning of the RING-domain (∆aa 488-619). See also Fig. EV3B.


( B ) Euc1 binds Slx5 in vitro . Recombinant purified GST or GSTEuc1 were used in GST pull - down assays to co - precipitate recombinant 6HisSlx5 .

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(B) Euc1 binds Slx5 in vitro. Recombinant purified GST or GSTEuc1 were used in GST pull-down assays to co-precipitate recombinant 6HisSlx5.