E , F . The relative m6A and YTHDF1 enrichment of the BZLF1 ( E ) or BRLF1 ( F ) mRNAs measured in CNE2EBV cells transfected with the METTL14 - specific siRNAs ( siM14 ) or siNC control following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h , followed by MeRIP and RIP assays . The fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample .

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

E, F. The relative m6A and YTHDF1 enrichment of the BZLF1 (E) or BRLF1 (F) mRNAs measured in CNE2EBV cells transfected with the METTL14-specific siRNAs (siM14) or siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h, followed by MeRIP and RIP assays. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


D . The interaction between YTHDF1 and DCP2 was detected by co - IP in 293T cells .

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D. The interaction between YTHDF1 and DCP2 was detected by co-IP in 293T cells.


F . The interactions between endogenous YTHDF1 and ZAP , DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2 - , YTHDF3 - specific siRNAs or the siNC control . YTHDF1 , YTHDF2 , YTHDF3 , ZAP , DDX17 , and DCP2 were detected in the immunoprecipitated complexes using specific antibodies , respectively .

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

F. The interactions between endogenous YTHDF1 and ZAP, DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2-, YTHDF3-specific siRNAs or the siNC control. YTHDF1, YTHDF2, YTHDF3, ZAP, DDX17, and DCP2 were detected in the immunoprecipitated complexes using specific antibodies, respectively.


F . The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1 - specific siRNAs and XRN1 - specific siRNAs . The C666 cells were co - transfected with YTHDF1 - specific siRNAs and XRN1 - specific siRNAs for 24 h . The mRNA levels were determined using qPCR .

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F. The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs. The C666 cells were co-transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs for 24 h. The mRNA levels were determined using qPCR.


G . The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP , DDX17 , or DCP2 in CNE2EBV cells following EBV reactivation . CNE2EBV cells were transfected with plasmids encoding myc - ZAP , myc - DDX17 , myc - DCP2 , or vector control . At 24 h post transfection , the cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h . The RIP assays were performed using Myc - beads . The fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP, DDX17, or DCP2 in CNE2EBV cells following EBV reactivation. CNE2EBV cells were transfected with plasmids encoding myc-ZAP, myc-DDX17, myc-DCP2, or vector control. At 24 h post transfection, the cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using Myc-beads. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


H . Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1 - specific siRNA or the siNC control . After 24 h of siRNA transfection , HK1 cells were infected with EBV and analyzed by FACS at 24 hpi .

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

H. Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1-specific siRNA or the siNC control. After 24 h of siRNA transfection, HK1 cells were infected with EBV and analyzed by FACS at 24 hpi.


B . Volcano plot of differently - regulated phosphosites of MAPK cascade components from 3DIV cultured embryonic DRG neurons upon 4h of NGF deprivation , with protein - level cutoffs set at log2 fold change > 1 . 0 and - log10 adjusted P value > 1 . 3 ( P < 0 . 05 ) , marked by dashed lines . Downregulated sites represented as blue points , and up - regulated sites as orange points ( n = 2 independent experiments ) .

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B. Volcano plot of differently-regulated phosphosites of MAPK cascade components from 3DIV cultured embryonic DRG neurons upon 4h of NGF deprivation, with protein-level cutoffs set at log2 fold change >1.0 and -log10 adjusted P value >1.3 (P< 0.05), marked by dashed lines. Downregulated sites represented as blue points, and up-regulated sites as orange points (n = 2 independent experiments).


C . Representative Western blots of the different MAPKs expressed in DRG neurons ( DIV3 ) cultured in the presence or absence of NGF for 3h and subjected to a membrane / cytosolic fractionation . α - tubulin and STMN - 1 are used as controls of cytosolic proteins and Na + / K + - ATPase and STMN - 2 as controls of membrane proteins .

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C. Representative Western blots of the different MAPKs expressed in DRG neurons (DIV3) cultured in the presence or absence of NGF for 3h and subjected to a membrane/cytosolic fractionation. α-tubulin and STMN-1 are used as controls of cytosolic proteins and Na+/K+-ATPase and STMN-2 as controls of membrane proteins.


D . Quantification of the ratio of protein localized in the membrane fraction after 3h of NGF deprivation ( n = 6 - 14 biological replicates ; series of unpaired t tests followed by a Holm - Sidak correction ) . Data information : Graphs represent mean ± SEM in ( D ) * p < 0 . 05 and * * p < 0 . 01 .

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D. Quantification of the ratio of protein localized in the membrane fraction after 3h of NGF deprivation (n = 6-14 biological replicates; series of unpaired t tests followed by a Holm-Sidak correction). Data information: Graphs represent mean ± SEM in (D) *p < 0.05 and **p < 0.01.


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 .

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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 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 )

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

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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 ) , 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 )

O O O O O O O O O O O O O O O O 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 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 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 )

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

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 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 )

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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 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 .

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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 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 )

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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 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 .

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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 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 )

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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)


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 .

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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 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 ) .

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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 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 .

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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 ) 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 )

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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 ) 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 )

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

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 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 ) .

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

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 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 .

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

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

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 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 .

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

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 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 .

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

E-F. 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 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 .

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

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

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 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 )

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

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 and Na + / K + - ATPase from cytosolic , membrane and 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 O O O O O 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 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 .

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

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 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 .

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

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 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

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)


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 .

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

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 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-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

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 ) 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 ) .

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

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 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 .

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 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 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

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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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-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

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 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 .

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

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

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 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 ) .

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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 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 )

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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 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 )

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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 , 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 .

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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 cells ( mHEVc ; B ) and human retinal pigment epithelial cells ( 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 . Cell 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 . 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 .

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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 ± 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 .

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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 . 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 .

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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 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 .

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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 . Cells 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 light microscopy 2 h and 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 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 .

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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 ) 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 .

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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 fluorescent microscopy 8 h post - challenge . Arrowheads indicate accumulation of LAMP - 1 around the

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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 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 .

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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 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 .

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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 the mean ± SEM and 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 . 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

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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 . Cell lysates were used to examine total Akt and 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 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 .

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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 and 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 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 .

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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 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 .

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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 . 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 .

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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 light microscopy at 2 and 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 cells transfected with either EGFR or control siRNA were challenged with T . gondii followed by examination of monolayers by light 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 and challenged with T . gondii . Monolayers were examined by light microscopy at 2 and 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 and CHO - EGFR cells 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 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 .

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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 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 .

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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 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 .

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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 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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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 cells ( 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).


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 .

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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 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 .

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

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 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 .

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

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 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 .

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

. 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 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 .

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

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 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 .

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 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 O O O O O O O O O O

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.


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

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

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


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 .

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

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 ) 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 - tRNA < sub > UU < em > C < / em > < / sub > < sup > Gl < em > u < / em > < / sup > ; 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 .

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

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 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 .

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

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.


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 .

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

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.


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

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

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 analysis of ScUba4 mutant yeast strains in response to rapamycin .

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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

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 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 .

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

(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 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 .

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

(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 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 ) .

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

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

(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 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 ) .

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

(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 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 ) .

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

(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 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 .

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

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 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 .

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

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 the indicated strains and enriched DNA was analyzed by qPCR . Data represent means ± SD ( n = 4 ) .

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

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 .

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

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 .

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

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 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 .

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

(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.