Figure 6A - D High - resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient ( − / − , black bars ) or not ( + / + , white bars ) using glucose ( A ) , lipid - related substrates ( C ) or complex IV substrate ( D ) . High - resolution respirometry was also performed on intact MEFi ( B ) . For different respiratory state details ( routine , 3 , leak , and ETS capacity ) , please refer to the Materials and Methods section . Results are expressed as the mean ± SEM and are representative of three independent experiments . In ( A ) : P ( − / − versus + / + ; State 3 ) = 0 . 0079 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( B ) : P ( − / − versus + / + ; routine ) = 0 . 0159 ; P ( − / − versus + / + ; leak ) = 0 . 0159 ; P ( − / − versus + / + ; ETS ) = 0 . 0159 ; n = 5 in each group . In ( C ) : P ( − / − versus + / + ; State 3 ) = 0 . 029 ; P ( − / − versus + / + ; ETS ) = 0 . 0079 ; n = 5 in each group . In ( D ) : P ( − / − versus + / + ; ETS ) = 0 . 0259 ; n = 5 in each group . * TP53INP1 − / − versus TP53INP1 + / + ; 1 character : P < 0 . 05 ; 2 characters : P < 0 . 01 . E TCLs from MEFi were analyzed by immunoblotting for CPT1A , CPT1B and β - tubulin . Results are representative of three independent experiments .

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

Figure 6A-D High-resolution respirometry was performed on permeabilized MEFi TP53INP1 deficient (−/−, black bars) or not (+/+, white bars) using glucose (A), lipid-related substrates (C) or complex IV substrate (D). High-resolution respirometry was also performed on intact MEFi (B). For different respiratory state details (routine, 3, leak, and ETS capacity), please refer to the Materials and Methods section. Results are expressed as the mean ± SEM and are representative of three independent experiments. In (A): P (−/− versus +/+; State 3) = 0.0079; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (B): P (−/− versus +/+; routine) = 0.0159; P (−/− versus +/+; leak) = 0.0159; P (−/− versus +/+; ETS) = 0.0159; n = 5 in each group. In (C): P (−/− versus +/+; State 3) = 0.029; P (−/− versus +/+; ETS) = 0.0079; n = 5 in each group. In (D): P (−/− versus +/+; ETS) = 0.0259; n = 5 in each group. *TP53INP1−/− versus TP53INP1 +/+; 1 character: P < 0.05; 2 characters: P < 0.01.E TCLs from MEFi were analyzed by immunoblotting for CPT1A, CPT1B and β-tubulin. Results are representative of three independent experiments.


Figure 7MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were seeded in media containing or not 10 mM NAC . Forty - eight hours later , MEFi were treated or not ( NT ) with 100 μM hydrogen peroxide ( H2O2 ) during 1 h in serum free media . Cells were left to recover for 4 h in normal media containing or not NAC ( 10 mM ) , GW - 9662 ( PPARγ inhibitor , 10 μM ) or PNU - 74654 ( Wnt / β - catenin pathway inhibitor , 50 μM ) before being harvested . Cells were observed by TEM . Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure . Scale bar represents 0 . 5 μm . Results are expressed as the mean ± SEM and are representative of three independent experiments . * P ( − / − versus + / + ; NT ) = 0 . 00086 ; # P ( H2O2 versus NT ; + / + ) = 0 . 013 . Cells were also observed by fluorescence microscopy after Bodipy493 / 503 staining of LD ( green ) and DAPI staining of nucleus ( blue ) . L = lipid droplets . Scale bar represents 100 μm . Results are representative of three independent experiments . TCLs from MEFi were analyzed by immunoblotting for β - catenin , PPARγ , ATGL , MGLL and β - tubulin . Results are representative of three independent experiments .

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

Figure 7MEFi deficient (−/−) or not (+/+) for TP53INP1 were seeded in media containing or not 10 mM NAC. Forty-eight hours later, MEFi were treated or not (NT) with 100 μM hydrogen peroxide (H2O2) during 1 h in serum free media. Cells were left to recover for 4 h in normal media containing or not NAC (10 mM), GW-9662 (PPARγ inhibitor, 10 μM) or PNU-74654 (Wnt/β-catenin pathway inhibitor, 50 μM) before being harvested. Cells were observed by TEM.Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure. Scale bar represents 0.5 μm. Results are expressed as the mean ± SEM and are representative of three independent experiments. *P (−/− versus +/+; NT) = 0.00086; #P (H2O2 versus NT; +/+) = 0.013.Cells were also observed by fluorescence microscopy after Bodipy493/503 staining of LD (green) and DAPI staining of nucleus (blue). L = lipid droplets. Scale bar represents 100 μm. Results are representative of three independent experiments.TCLs from MEFi were analyzed by immunoblotting for β-catenin, PPARγ, ATGL, MGLL and β-tubulin. Results are representative of three independent experiments.


Figure 2 ( A ) Volcano plot displaying the associations between the mutational status of genes and the activity of kinases . The x - axis contains the mutation coefficients ( effect sizes ) and the y - axis the adjusted P - values . The vertical line in the x - axis is at x = 0 and separates the positive ( right ) from the negative ( left ) associations . The horizontal lines in the y - axis represent the points of statistical significance in the plot ( adjusted P - values < 0 . 05 and < 0 . 01 ) . The associations are represented in the form of a mutated gene - kinase . The colour gradient represents the string network edge weight interval of the pair ( grey if the pair is not in the string network ) . ( B ) Same as ( A ) for the TFs . The inner plot shows the effects of TF mutations on their own activities . ( ( C ) ( D ) Examples of the genetic associations highlighted in the volcano plots . The x - axis represents the associations and the y - axis the protein activities . The colours stratify the samples by their mutational status in the respective genes . The outliers ( defined as the data points beyond Q1 - 1 . 5 * IQR and Q3 + 1 . 5 * IQR , where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range ) were removed from the distributions for representation purposes . The number of protein activity quantifications ( including outliers ) are shown beneath each boxplot . The P - values from Wilcoxon rank sum tests comparing both distributions are shown . All data points ( including outliers ) were used to calculate the P - values .

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

Figure 2(A) Volcano plot displaying the associations between the mutational status of genes and the activity of kinases. The x-axis contains the mutation coefficients (effect sizes) and the y-axis the adjusted P-values. The vertical line in the x-axis is at x=0 and separates the positive (right) from the negative (left) associations. The horizontal lines in the y-axis represent the points of statistical significance in the plot (adjusted P-values < 0.05 and < 0.01).The associations are represented in the form of a mutated gene - kinase. The colour gradient represents the string network edge weight interval of the pair (grey if the pair is not in the string network). (B) Same as (A) for the TFs. The inner plot shows the effects of TF mutations on their own activities. ((C) (D) Examples of the genetic associations highlighted in the volcano plots. The x-axis represents the associations and the y-axis the protein activities. The colours stratify the samples by their mutational status in the respective genes. The outliers (defined as the data points beyond Q1-1.5*IQR and Q3+1.5*IQR, where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range) were removed from the distributions for representation purposes. The number of protein activity quantifications (including outliers) are shown beneath each boxplot. The P-values from Wilcoxon rank sum tests comparing both distributions are shown. All data points (including outliers) were used to calculate the P-values.


Figure 3 ( C ) Kinase activity gradient along the samples for a selection of the kinases shown in ( B ) . ( F ) Linear regression between the percentage of samples where the kinase is regulated in the perturbed conditions and in the tumour samples . The trend line and the Pearson ' s r , with the respective P - value , are shown . In red and green are the kinases preferably regulated in the tumours and in the conditions , respectively . In blue are the kinases regulated in both . The 95 % confidence interval around the regression line is represented by the shaded area .

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

Figure 3(C) Kinase activity gradient along the samples for a selection of the kinases shown in (B).(F) Linear regression between the percentage of samples where the kinase is regulated in the perturbed conditions and in the tumour samples. The trend line and the Pearson's r, with the respective P-value, are shown. In red and green are the kinases preferably regulated in the tumours and in the conditions, respectively. In blue are the kinases regulated in both. The 95% confidence interval around the regression line is represented by the shaded area.


Figure 4 ( C - F ) Kaplan - Meier survival plots comparing the survival probabilities ( y - axes ) as a function of time in days ( x - axes ) for proteins and cancer types shown in ( A ) and ( B ) . ( C ) MYC / brain cancer ( inactive = 71 , neutral = 39 , active = 67 ) . ( D ) MYC / liver cancer ( inactive = 58 , neutral = 59 , active = 40 ) . ( E ) MAP3K8 / renal cancer ( neutral = 95 , active = 7 ) . ( F ) PRKCA / renal cancer ( inactive = 3 , neutral = 92 , active = 7 ) . The log - rank P - values are shown in the plots .

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

Figure 4(C-F) Kaplan-Meier survival plots comparing the survival probabilities (y-axes) as a function of time in days (x-axes) for proteins and cancer types shown in (A) and (B). (C) MYC/brain cancer (inactive = 71, neutral = 39, active = 67). (D) MYC/liver cancer (inactive = 58, neutral = 59, active = 40). (E) MAP3K8/renal cancer (neutral = 95, active = 7). (F) PRKCA/renal cancer (inactive = 3, neutral = 92, active = 7). The log-rank P-values are shown in the plots.


Figure 1 ( C ) Time - lapse microscopy of cells containing a PsigV - YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress . The stress ( red line ) was added between the 200 mins and 300 min time points ( at 240 min ) . The red arrow highlights a cell with a delayed activation of σV . Scale bar : 5 μm .

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Figure 1(C) Time-lapse microscopy of cells containing a PsigV-YFP promoter reporter reveals heterogeneous activation of σV in response to lysozyme stress. The stress (red line) was added between the 200 mins and 300 min time points (at 240 min). The red arrow highlights a cell with a delayed activation of σV. Scale bar: 5 μm.


Figure 2 ( B ) A priming ( 30 min ) stress of 1 µg / ml lysozyme followed by the high lysozyme stress ( 20 µg / ml ) improves survival . The solid blue lines are the biological repeats for the no priming experiment ( n = 2 ) whereas the solid red lines are the biological repeats for the priming experiment ( n = 4 ) . ( Total number of cells shown for No Priming , N = 2013 and Priming , N = 4937 ) .

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Figure 2(B) A priming (30 min) stress of 1 µg/ml lysozyme followed by the high lysozyme stress (20 µg/ml) improves survival. The solid blue lines are the biological repeats for the no priming experiment (n=2) whereas the solid red lines are the biological repeats for the priming experiment (n=4). (Total number of cells shown for No Priming, N = 2013 and Priming, N = 4937).


Figure 3 ( B , C ) RNA - seq experiment on WT ( JLB130 ) and ΔsigV ( JLB154 ) strains , showing quantification of the absolute expression of individual genes in the presence and absence of lysozyme stress . The shaded grey box represents a ± 5 fold change . DEseq was used to identify genes which were differentially expressed with a 5 % p - value cutoff between the WT and ΔsigV mutant in response to lysozyme treatment ( default DEseq test was used ) . ( B ) Only the sigV operon is strongly activated ( > 5 fold change ) in response to lysozyme stress in WT ( JLB130 ) , as previously reported ( Guariglia - Oropeza and Helmann 2011 ) . ( C ) No genes were strongly upregulated in ΔsigV ( JLB154 ) by lysozyme stress . ( D ) Effect of the overexpression of individual components of the σV pathway ( Biological repeats : WT : n = 8 , sigV + : n = 4 , rsiV + : n = 3 oatA + : n = 4 , yrhK + : n = 3 , sipS + : n = 3 and rasP + : n = 3 , pbpX + : n = 6 ) on the fraction of activated cells . Only overexpression of sigV , rsiV or oatA changed the observed dynamics compared to WT . The histograms of the shown data are shown in Appendix Figure S16 . ( E ) Deleting oatA did not alter the σV activation dynamics . However , deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug / ml lysozyme . n > = 3 biological repeats for all data shown . The histograms of the shown data are shown in Appendix Figure S17 . (

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

Figure 3(B, C) RNA-seq experiment on WT (JLB130) and ΔsigV (JLB154) strains, showing quantification of the absolute expression of individual genes in the presence and absence of lysozyme stress. The shaded grey box represents a ± 5 fold change. DEseq was used to identify genes which were differentially expressed with a 5% p-value cutoff between the WT and ΔsigV mutant in response to lysozyme treatment (default DEseq test was used). (B) Only the sigV operon is strongly activated (>5 fold change) in response to lysozyme stress in WT (JLB130), as previously reported (Guariglia-Oropeza and Helmann 2011). (C) No genes were strongly upregulated in ΔsigV (JLB154) by lysozyme stress.(D) Effect of the overexpression of individual components of the σV pathway (Biological repeats: WT: n=8, sigV+: n=4, rsiV+: n=3 oatA+: n=4, yrhK+: n=3, sipS+: n=3 and rasP+: n=3, pbpX+: n=6) on the fraction of activated cells. Only overexpression of sigV, rsiV or oatA changed the observed dynamics compared to WT. The histograms of the shown data are shown in Appendix Figure S16. (E) Deleting oatA did not alter the σV activation dynamics. However, deleting sigV or rsiV resulted in no further activation of σV in response to 1 ug/ml lysozyme. n>=3 biological repeats for all data shown. The histograms of the shown data are shown in Appendix Figure S17. (


Figure 5 ( C ) As predicted , the 2xsigV or 2xsigV - rsiV stains have a homogenous σV response to 1 µg / ml lysozyme , while the 2xrsiV strain has increased heterogeneity . Each bar plot is the average of three biological repeats . The bars correspond to the mean ± s . d . .

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

Figure 5(C) As predicted, the 2xsigV or 2xsigV-rsiV stains have a homogenous σV response to 1 µg/ml lysozyme, while the 2xrsiV strain has increased heterogeneity. Each bar plot is the average of three biological repeats. The bars correspond to the mean ± s.d..


Figure 6 ( E , F ) For very long intervals between stress ( 12h ) , the heterogeneity is the same as if the cells had not experienced a prior lysozyme stress ( control ) . N > 50 single - cell traces .

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Figure 6(E, F) For very long intervals between stress (12h), the heterogeneity is the same as if the cells had not experienced a prior lysozyme stress (control). N>50 single-cell traces.


Figure 1B . 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 . 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 .

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

Figure 1B. 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.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.


Figure 2A . 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 ) . 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 ) 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 . 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 .

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

Figure 2A. 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).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)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.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.


Figure 3Representative 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Representative 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.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.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)


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

Figure 4A. 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.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.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.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)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.


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

Figure 5A. 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.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.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).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.


Figure 6A . 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 . 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 ) . 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 )

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

Figure 6A. 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.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).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)


Figure 1Western blot analysis of Fxr1 during ( A ) , TTX ( 48h treatment ) inducedupscaling ( n = 6 in each condition ) of primary postnatal cortical cultures . Student ' s T - test * p < 0 . 05 , * * * p < 0 . 001 . Western blot analysis of Fxr1 during ( B ) , BIC ( 48h treatment ) induced downscaling ( n = 8 in each condition ) of primary postnatal cortical cultures . Student ' s T - test * p < 0 . 05 , * * * p < 0 . 001 . C , D , Western blot analysis of ( C ) , Fxr2 ( Veh n = 8 , TTX n = 8 ) and ( D ) , Fmrp ( Veh n = 10 , TTX n = 12 ) during upscaling , Student ' s T - test * p < 0 . 05 . E - G , RT - qPCR measurement of mRNA for ( E ) , Fxr1 , ( F ) , Fxr2 , ( G ) , Fmr1 during upscaling . n = 4 in each condition , Student ' s T - test * p < 0 . 05 . H , Western blot analysis for Fxr1 , pGsk3α / β , Gsk3α / β and GAPDH in neuronal cultures treated with 1µM TTX ( TTX ) or 1mM LiCI ( Li ) or 1mM NaCI ( Ctrl ) for 48h . I , Expression of Fxr1 protein in TTX ( n = 5 ) and Li ( n = 5 ) conditions relative to Ctrl ( n = 6 ) condition . One way ANOVA with Dunnett ' s Multiple Comparison Test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . J , Expression of pGsk3β / Gsk3β or pGsk3α / Gsk3α in TTX ( n = 5 ) and Li ( n = 5 ) conditions relative to Ctrl ( n = 6 ) condition . One way ANOVA with Dunnett ' s Multiple Comparison Test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 1Western blot analysis of Fxr1 during (A), TTX (48h treatment) inducedupscaling (n=6 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.Western blot analysis of Fxr1 during (B), BIC (48h treatment) induced downscaling (n=8 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.C, D, Western blot analysis of (C), Fxr2 (Veh n=8, TTX n=8) and (D), Fmrp (Veh n=10, TTX n=12) during upscaling, Student's T-test *p<0.05.E-G, RT-qPCR measurement of mRNA for (E), Fxr1, (F), Fxr2, (G), Fmr1 during upscaling. n=4 in each condition, Student's T-test *p<0.05.H, Western blot analysis for Fxr1, pGsk3α/β, Gsk3α/β and GAPDH in neuronal cultures treated with 1µM TTX (TTX) or 1mM LiCI (Li) or 1mM NaCI (Ctrl) for 48h.I, Expression of Fxr1 protein in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.J, Expression of pGsk3β/Gsk3β or pGsk3α/Gsk3α in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


Figure 2B , Western blot analysis of total GluA1 expression in Ctrl or Fxr1 overexpression ( Fxr1 ) condition during upscaling ( Ctrl / Veh n = 5 , Ctrl / TTX n = 5 , Fxr1 / Veh n = 6 , Fxr1 / Veh n = 6 ) . One way Anova with Dunnett ' s Multiple Comparison Test * * * p < 0 . 001 . C , Western blot analysis of total GluA1 expression in Ctrl or Fxr1over condition during downscaling ( Ctrl / Veh n = 4 , Ctrl / TTX n = 4 , Fxr1 / Veh n = 4 , Fxr1 / Veh n = 4 ) . D , RT - qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling ( Ctrl / Veh n = 3 , Ctrl / TTX n = 3 , Fxr1 / Veh n = 3 , Fxr1 / Veh n = 3 ) . One way Anova with Bonferroni ' s Multiple Comparison Test * * * p < 0 . 001 . F , Measurement of relative luciferase signal after co - transfection of different tagged luciferase constructs along with GFP ( Ctrl ) or GFP - Fxr1 ( Fxr1 ) plasmids . n = 5 in each condition , Student ' s T - test * p < 0 . 05 , * * * p < 0 . 001 . G , Immunostaining for surface GluA1 in GFP ( Ctrl ) or GFP - Fxr1 ( Fxr1 over ) infected cultures after treatment with Veh , TTX or BIC for 48h . H , Percentage of surface GluA1 relative to the mean of Ctrl / Veh condition ( Ctrl condition : Veh n = 63 , TTX n = 34 , BIC n = 29 , Fxr1 condition : Veh n = 56 , TTX n = 30 , BIC n = 29 ) . One way Anova with Bonferroni ' s Multiple Comparison Test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 2B, Western blot analysis of total GluA1 expression in Ctrl or Fxr1 overexpression (Fxr1) condition during upscaling (Ctrl/Veh n=5, Ctrl/TTX n=5, Fxr1/Veh n=6, Fxr1/Veh n=6). One way Anova with Dunnett's Multiple Comparison Test ***p<0.001.C, Western blot analysis of total GluA1 expression in Ctrl or Fxr1over condition during downscaling (Ctrl/Veh n=4, Ctrl/TTX n=4, Fxr1/Veh n=4, Fxr1/Veh n=4).D, RT-qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling (Ctrl/Veh n=3, Ctrl/TTX n=3, Fxr1/Veh n=3, Fxr1/Veh n=3). One way Anova with Bonferroni's Multiple Comparison Test ***p<0.001.F, Measurement of relative luciferase signal after co-transfection of different tagged luciferase constructs along with GFP (Ctrl) or GFP-Fxr1 (Fxr1) plasmids. n=5 in each condition, Student's T-test *p<0.05, ***p<0.001.G, Immunostaining for surface GluA1 in GFP (Ctrl) or GFP-Fxr1 (Fxr1 over) infected cultures after treatment with Veh, TTX or BIC for 48h.H, Percentage of surface GluA1 relative to the mean of Ctrl/Veh condition (Ctrl condition: Veh n=63, TTX n=34, BIC n=29, Fxr1 condition: Veh n=56, TTX n=30, BIC n=29 ). One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


Figure 3A , Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure ( Ctrl / Veh n = 16 and Ctrl / TTX n = 17 ) . B , A linear fit of Ctrl / TTX and Ctrl / Veh amplitudes . C , The degrees of overlap between Ctrl / TTX and Ctrl / Veh data were assessed using various scaling factors . The largest nonsignificant p - value was obtained with 1 . 47 scaling factor . D , Cumulative probability plots of the mEPSCs amplitude of Ctrl / Veh , Ctrl / TTX and Ctrl / TTX divided by scaling factor 1 . 47 , which yielded the maximum overlap with Ctrl / Veh data . Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( E ) Fxr1overexpressing neurons ( Fxr1 / Veh n = 10 and Fxr1 / TTX n = 8 ) Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( F ) Gsk3 KO neurons ( Gsk3KO / Veh n = 10 and Gsk3KO / TTX n = 11 ) Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( G ) Fxr1 KO neurons ( Fxr1KO / Veh n = 16 and Fxr1KO / TTX n = 8 ) . H , A linear fit of Fxr1 KO / Veh and Ctrl / Veh amplitudes . I , The degrees of overlap between Fxr1 KO / Veh and Ctrl / Veh data were assessed using various scaling factors . The largest non - significant p - value was obtained with 1 . 27 scaling factor . J , Cumulative probability plots of the mEPSCs amplitude of Ctrl / Veh , Fxr1 KO / Veh and Fxr1 KO / Veh divided by scaling factor 1 . 27 , which yielded the maximum overlap with Ctrl / Veh data . K , Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from cultured cortical Gsk3 and Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure ( Gsk3 / Fxr1KO / Veh n = 8 and Gsk3 / Fxr1KO / TTX n = 11 ) . L , A linear fit of Gsk3 / Fxr1 KO / Veh and Ctrl / Veh amplitudes . M , The degrees of overlap between Gsk3 / Fxr1 KO / Veh and Ctrl / Veh data were assessed using various scaling factors . The largest non - significant p - value was obtained with 1 . 68 scaling factor . N , Cumulative probability plots of the mEPSCs amplitude of Ctrl / Veh , Gsk3 / Fxr1 KO / Veh and Gsk3 / Fxr1 KO / Veh divided by scaling factor 1 . 68 , which yielded the maximum overlap with Ctrl / Veh data . O , mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure . One way Anova with Bonferroni ' s Multiple Comparison Test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . P , mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure .

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

Figure 3A, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure (Ctrl/Veh n=16 and Ctrl/TTX n=17). B, A linear fit of Ctrl/TTX and Ctrl/Veh amplitudes. C, The degrees of overlap between Ctrl/TTX and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant p-value was obtained with 1.47 scaling factor. D, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Ctrl/TTX and Ctrl/TTX divided by scaling factor 1.47, which yielded the maximum overlap with Ctrl/Veh data. Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (E) Fxr1overexpressing neurons (Fxr1/Veh n=10 and Fxr1/TTX n=8)Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (F) Gsk3 KO neurons (Gsk3KO/Veh n=10 and Gsk3KO/TTX n=11)Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (G) Fxr1 KO neurons (Fxr1KO/Veh n=16 and Fxr1KO/TTX n=8). H, A linear fit of Fxr1 KO/Veh and Ctrl/Veh amplitudes. I, The degrees of overlap between Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.27 scaling factor. J, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Fxr1 KO/Veh and Fxr1 KO/Veh divided by scaling factor 1.27, which yielded the maximum overlap with Ctrl/Veh data. K, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical Gsk3 and Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure (Gsk3/Fxr1KO/Veh n=8 and Gsk3/Fxr1KO/TTX n=11). L, A linear fit of Gsk3/Fxr1 KO/Veh and Ctrl/Veh amplitudes. M, The degrees of overlap between Gsk3/Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.68 scaling factor. N, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Gsk3/Fxr1 KO/Veh and Gsk3/Fxr1 KO/Veh divided by scaling factor 1.68, which yielded the maximum overlap with Ctrl/Veh data. O, mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure. One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.P, mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure.


Figure 4A , Hourly distribution of wakefulness ( WAKE ) , slow - wave sleep ( SWS ) and paradoxical sleep ( PS ) during a 24 - h baseline ( BL ) recording and a second 24 - h starting with 6 - h sleep deprivation ( SD ) ( recovery : REC ) in Ctrl and Fxr1 overexpressing ( Fxr1 ) mice . Significant Group - by - Hour interactions were found for wakefulness during BL ( F23 , 230 = 1 . 67 , p = 0 . 046 ) and REC ( F23 , 230 = 2 . 83 , p = 0 . 0029 ) , for SWS during BL ( F23 , 230 = 1 . 66 , p = 0 . 045 ) and REC ( F23 , 230 = 2 . 89 , p = 0 . 0028 ) , and for PS during REC ( F16 , 160 = 2 . 54 , p = 0 . 0018 , Two - way ANOVA Huynh - Feldt corrected , * p < 0 . 05 ) . B , Power spectra for WAKE , SWS , and PS in Ctrl and Fxr1 mice computed between 0 . 75 and 50 Hz per 0 . 25 - Hz for the full 24 - h of BL and REC . C , D , The spectral activity of Fxr1 mice expressed relative to that of Ctrl mice for WAKE , SWS and PS during ( C ) the 24 - h BL and ( D ) the 24 - h REC . A significant difference between groups was found for the frequency band 8 to 11 . 75 Hz during wakefulness ( t = 2 . 30 , p = 0 . 044 Student ' s T - test * p < 0 . 05 ) . E , Time course of wakefulness spectral activity ratio between low alpha ( 8 . 5 - 10 . 5 Hz ) and low theta ( 4 - 6 Hz ) during BL and REC in Ctrl and Fxr1 mice . A significant Group - by - Interval interaction was found for REC ( F22 , 220 = 1 . 96 , Two - way ANOVA Huynh - Feldt corrected ; * p < 0 . 05 ) , and also when using BL and REC intervals of the light periods ( F16 , 160 = 3 . 11 , p < 0 . 01 ) , . F , Time course of SWS relative delta activity during BL and REC in Ctrl and Fxr1 mice . A significant Group - by - Interval interaction was found during REC ( F13 , 130 = 1 . 89 , Two - way ANOVA Huynh - Feldt corrected ; * p < 0 . 05 ) .

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

Figure 4A, Hourly distribution of wakefulness (WAKE), slow-wave sleep (SWS) and paradoxical sleep (PS) during a 24-h baseline (BL) recording and a second 24-h starting with 6-h sleep deprivation (SD) (recovery: REC) in Ctrl and Fxr1 overexpressing (Fxr1) mice. Significant Group-by-Hour interactions were found for wakefulness during BL (F23,230 = 1.67, p = 0.046) and REC (F23,230 = 2.83, p = 0.0029), for SWS during BL (F23,230 = 1.66, p = 0.045) and REC (F23,230 = 2.89, p=0.0028), and for PS during REC (F16,160 = 2.54, p=0.0018, Two-way ANOVA Huynh-Feldt corrected, *p<0.05).B, Power spectra for WAKE, SWS, and PS in Ctrl and Fxr1 mice computed between 0.75 and 50 Hz per 0.25-Hz for the full 24-h of BL and REC.C, D, The spectral activity of Fxr1 mice expressed relative to that of Ctrl mice for WAKE, SWS and PS during (C) the 24-h BL and (D) the 24-h REC. A significant difference between groups was found for the frequency band 8 to 11.75 Hz during wakefulness (t = 2.30, p=0.044 Student's T-test *p<0.05).E, Time course of wakefulness spectral activity ratio between low alpha (8.5-10.5 Hz) and low theta (4-6 Hz) during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found for REC (F22,220 = 1.96, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05), and also when using BL and REC intervals of the light periods (F16,160 = 3.11, p < 0.01),.F, Time course of SWS relative delta activity during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found during REC (F13,130 = 1.89, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05).


Figure 5Western blot analysis of ( B ) p845GluA1 and GluA1 expression ( S n = 9 , SD n = 10 ) in the prefrontal cortex of sleeping and sleep deprived mice . Student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Western blot analysis of , ( C ) p880GluA2 and GluA2 expression ( S n = 9 , SD n = 8 ) in the prefrontal cortex of sleeping and sleep deprived mice . Student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Western blot analysis of ( D ) Fxr1 expression ( S n = 11 , SD n = 10 ) in the prefrontal cortex of sleeping and sleep deprived mice . Student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Western blot analysis of ( E ) Fmrp expression ( S n = 11 , SD n = 10 ) in the prefrontal cortex of sleeping and sleep deprived mice . Student ' s t - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . F - H , RNAseq measurement of mRNA for ( F ) Fxr1 , ( G ) Fxr2 , ( H ) Fmr1 during SD . n = 3 in each condition , Student ' s T - test * p < 0 . 05 .

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

Figure 5Western blot analysis of (B) p845GluA1 and GluA1 expression (S n=9, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.Western blot analysis of , (C) p880GluA2 and GluA2 expression (S n=9, SD n=8) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.Western blot analysis of (D) Fxr1 expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.Western blot analysis of (E) Fmrp expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.F-H, RNAseq measurement of mRNA for (F) Fxr1, (G) Fxr2, (H) Fmr1 during SD. n=3 in each condition, Student's T-test *p<0.05.


Figure 6Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from brain slices of S and SD mice . ( B ) Control neurons ( Ctrl / S n = 8 cells / 4 mice , Ctrl / SD n = 8 cells / 4 mice ) Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from brain slices of S and SD mice . ( C ) Fxr1 overexpressing ( Fxr1 / S n = 7 cells / 5 mice , Fxr1 over / SD n = 9 cells / 5 mice ) Cumulative probability plots of mEPSCs amplitude ( 500 events per cell ) and representative examples of mEPSCs ( left panel ) recorded from brain slices of S and SD mice . ( D ) Gsk3 sKO ( Gsk3sKO / S n = 6 cells / 3 mice , Gsk3sKO / SD n = 7 cells / 4 mice ) . mEPSC ( E ) mean amplitude of cortical neurons of S or SD mice . Control neurons ( Ctrl / S n = 8 cells / 4 mice , Ctrl / SD n = 8 cells / 4 mice ) , Fxr1 overexpressing ( Fxr1 / S n = 7 cells / 5 mice , Fxr1 over / SD n = 9 cells / 5 mice ) , and Gsk3 sKO ( Gsk3sKO / S n = 6 cells / 3 mice , Gsk3sKO / SD n = 7 cells / 4 mice ) . Student ' s T - test * p < 0 . 05 . F , mEPSC ( F ) frequency of cortical neurons of S or SD mice . Control neurons ( Ctrl / S n = 8 cells / 4 mice , Ctrl / SD n = 8 cells / 4 mice ) , Fxr1 overexpressing ( Fxr1 / S n = 7 cells / 5 mice , Fxr1 over / SD n = 9 cells / 5 mice ) , and Gsk3 sKO ( Gsk3sKO / S n = 6 cells / 3 mice , Gsk3sKO / SD n = 7 cells / 4 mice ) . Student ' s T - test * p < 0 . 05 . G , Representative examples of the current - voltage relationship of evoked EPSC amplitude recorded from S ( top panel ) and SD ( bottom panel ) mice . H , Summary bar graphs showing rectification index of control ( S n = 12 cells / 5 mice , and SD n = 10 cells / 5 mice ) , Fxr1P overexpressing ( S n = 9 cells / 4 mice , and SD n = 14 cells / 4 mice ) and Gsk3 sKO ( S n = 9 cells / 3 mice , and SD n = 9 cells / 3 mice ) neurons . One way ANOVA with Bonferroni ' s Multiple Comparison * p < 0 . 05 .

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Figure 6Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (B) Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice)Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (C) Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice)Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (D) Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice).mEPSC (E) mean amplitude of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05.F, mEPSC (F) frequency of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05.G, Representative examples of the current-voltage relationship of evoked EPSC amplitude recorded from S (top panel) and SD (bottom panel) mice.H, Summary bar graphs showing rectification index of control (S n=12 cells/5 mice, and SD n=10 cells/5 mice), Fxr1P overexpressing (S n=9 cells/4 mice, and SD n=14 cells/4 mice) and Gsk3 sKO (S n=9 cells/3 mice, and SD n=9 cells/3 mice) neurons. One way ANOVA with Bonferroni's Multiple Comparison *p < 0.05.


Figure 7B , Immunostaining for GFP and HA in Ctrl and Fxr1 mouse brain slices . Arrows indicate presence and arrowheads indicate an absence of GFP tagged Fxr1 granules . C , Quantification of colocalization of GFP and HA labeled neurons ( GFP + HA + 71 . 3 % ± 3 . 7 , n = 3 , GFP - Fxr1 + HA + 79 . 7 % ± 2 . 8 , n = 3 ) . G , Heat map showing transcripts that have bidirectional expression changes between Ctrl / S , Ctrl / SD , and Fxr1 / SD conditions .

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Figure 7B, Immunostaining for GFP and HA in Ctrl and Fxr1 mouse brain slices. Arrows indicate presence and arrowheads indicate an absence of GFP tagged Fxr1 granules.C, Quantification of colocalization of GFP and HA labeled neurons (GFP+HA+ 71.3%±3.7, n=3, GFP-Fxr1+HA+ 79.7%±2.8, n=3).G, Heat map showing transcripts that have bidirectional expression changes between Ctrl/S, Ctrl/SD, and Fxr1/SD conditions.


Figure 1A , B . RT - qPCR analysis of Zfp207 and Oct4 in mouse ESCs during ( A ) retinoic acid ( RA ) - induced and ( B ) embryoid body ( EB ) - mediated differentiation . Nestin was used as a neuronal differentiation marker to monitor RA - mediated differentiation . mRNA levels are relative to the expression at day 0 . J , K . ( J ) AP staining of shScr and Zfp207 - depleted ( sh1 and sh2 ) mouse ESCs . Scale bars , 20 µM . ( K ) Percentage of fully differentiated ( FD ) , partially differentiated ( PD ) and undifferentiated ( UN ) ESC colonies in shScr , sh1 and sh2 . M , N . ( M ) Western blot of ZFP207 , OCT4 , NANOG , and SOX2 in shScr , sh1 and sh2 ESCs and ( N ) RT - qPCR analysis of Oct4 , Nanog , and Sox2 in shScr , sh1 and sh2 ESCs ; data is relative to shScr . ( O ) Western blot of OCT4 during a 9 - h cycloheximide ( CHX ) time course treatment in shScr , sh1 and sh2 ESCs . ( Q ) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132 .

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Figure 1A, B. RT-qPCR analysis of Zfp207 and Oct4 in mouse ESCs during (A) retinoic acid (RA)-induced and (B) embryoid body (EB)-mediated differentiation. Nestin was used as a neuronal differentiation marker to monitor RA-mediated differentiation. mRNA levels are relative to the expression at day 0.J, K. (J) AP staining of shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Scale bars, 20 µM. (K) Percentage of fully differentiated (FD), partially differentiated (PD) and undifferentiated (UN) ESC colonies in shScr, sh1 and sh2.M, N. (M) Western blot of ZFP207, OCT4, NANOG, and SOX2 in shScr, sh1 and sh2 ESCs and (N) RT-qPCR analysis of Oct4, Nanog, and Sox2 in shScr, sh1 and sh2 ESCs; data is relative to shScr.(O) Western blot of OCT4 during a 9-h cycloheximide (CHX) time course treatment in shScr, sh1 and sh2 ESCs.(Q) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132.


Figure 2D . RT - qPCR of Zfp207 , the pluripotency genes ( Oct4 , Nanog and Sox2 ) , the mesodermal markers ( Msx1 and Brachyury , T ) , the endodermal markers ( Foxa2 , Sox17 ) , and the neural - associated genes ( Pax6 , Sox11 and Nestin ) in shScr , sh1 and sh2 ESCs along the time - course of EB - mediated differentiation . mRNA levels are relative to the expression of shScr at day 0 .

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Figure 2D. RT-qPCR of Zfp207, the pluripotency genes (Oct4, Nanog and Sox2), the mesodermal markers (Msx1 and Brachyury, T), the endodermal markers (Foxa2, Sox17), and the neural-associated genes (Pax6, Sox11 and Nestin) in shScr, sh1 and sh2 ESCs along the time-course of EB-mediated differentiation. mRNA levels are relative to the expression of shScr at day 0.


Figure 3B . Immunostaining of NESTIN ( green ) and TUJ1 ( red ) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation . Nuclei were counterstained with DAPI . Scale bar , 20 μM . ( E ) quantification of the signal in shScr , sh1 and sh2 at day 5 of neuroectodermal differentiation . G . RT - qPCR of Zfp207 and neural - associated markers in shScr , sh1 and sh2 along the course of neural differentiation . mRNA levels are relative to shScr at day 0 .

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Figure 3B. Immunostaining of NESTIN (green) and TUJ1 (red) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation. Nuclei were counterstained with DAPI. Scale bar, 20 μM.(E) quantification of the signal in shScr, sh1 and sh2 at day 5 of neuroectodermal differentiation.G. RT-qPCR of Zfp207 and neural-associated markers in shScr, sh1 and sh2 along the course of neural differentiation. mRNA levels are relative to shScr at day 0.


Figure 4 ( A ) Volcano plots of common differentially expressed genes in shScr and Zfp207 - depleted ( sh1 and sh2 ) mouse ESCs . Up - regulated ( Up ) and down - regulated ( Down ) genes are indicated in red and blue , respectively ( p < 0 . 05 ; > 1 . 5 - fold ) . Zfp207 is depicted in black . Grey dots indicate non - significant ( NS ) and < 1 . 5 - fold differential expressed genes .

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Figure 4(A) Volcano plots of common differentially expressed genes in shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Up-regulated (Up) and down-regulated (Down) genes are indicated in red and blue, respectively (p < 0.05; > 1.5-fold). Zfp207 is depicted in black. Grey dots indicate non-significant (NS) and < 1.5-fold differential expressed genes.


Figure 5A . Heatmap of column z - scores of log2 transformed values of genes involved in alternative splicing ( AS ) in shScr , sh1 and sh2 mouse ESCs . F . RT - PCR of Zfp207 - regulated AS events . For Mta1 , AS1 ( alternative splicing 1 ) and AS2 ( alternative splicing 2 ) . # 1 and # 2 indicate distinct biological replicates . * Denotes an isoform that was not taken in consideration for the quantification . The structure of each isoform is indicated ( not to scale ) . Alternative exons are blue . The percent spliced in ( PSI ) was quantified for each condition .

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Figure 5A. Heatmap of column z-scores of log2 transformed values of genes involved in alternative splicing (AS) in shScr, sh1 and sh2 mouse ESCs.F. RT-PCR of Zfp207-regulated AS events. For Mta1, AS1 (alternative splicing 1) and AS2 (alternative splicing 2). #1 and #2 indicate distinct biological replicates. *Denotes an isoform that was not taken in consideration for the quantification. The structure of each isoform is indicated (not to scale). Alternative exons are blue. The percent spliced in (PSI) was quantified for each condition.