Figure 6 ( A ) Developmental progression of Daxxm + z + , Daxxm - z + and Daxxm - z - early embryos . ( B ) Examples of morula and blastocyst embryos with normal and large polyploid nuclei . Data information : All scale bars 10µm . ( C ) Percentages of embryos with normal or polyploid nuclei . ( D ) Still images of time - lapse imaging of the first cleavage division in wild - type and maternally deficient zygotes expressing H2B - mCherry . Time points represent minutes after prometaphase . Lagging chromosomes and micronuclei are indicated by yellow arrowheads . Data information : All scale bars 10µm . ( E ) Percentage of zygotes with lagging chromosomes during 1st cleavage division . Data represent mean ± s . e . m . in n ≥ 3 replicate experiments . ( F ) IF of 5 - methyl and 5 - hydroxy - methyl cytosine staining of wild - type ( n = 42 ) , Daxxm - z + ( n = 14 ) and Ring1m - z + ; Rnf2m - z + ( n = 6 ) zygotes with lagging chromosomes . Yellow arrowheads indicate lagging chromosomes . Data information : All scale bars 10µm . ( G ) DNA - FISH for major ( green ) and minor ( red ) satellites on cleavage chromosomes of a Daxxm - z + zygote with a chromosome broken within the major satellite region . Data information : All scale bars 10µm . ( H ) H3 . 3 - EGFP live - imaging signal in pat - and mat - PCH of control Daxxm + z + ( n = 18 ) , Daxxm - z + ( n = 8 ) and Ring1m - z + ; Rnf2m - z + ( n = 12 ) zygotes . Scale bar 10µm . ( I ) Left : IF of endogenous H3 and ATRX at pat - PCH in control and Daxxm - z + PN5 zygotes . Right : Fluorescence intensity profiles of H3 and ATRX along white line in left panel at pat - PCH of zygotes . Scale bars 5 µm . Data information : All scale bars 10µm . ( J ) Boxplot displaying H3 signal intensity at pat - PCH of Daxxm + z + and Daxxm - z + PN5 zygotes . In boxplots , the enter lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by R software ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 times . * * * P < 0 . 001 , t - test , n = 12 for both groups . ( K ) IP of DAXX - EGFP and DAXXR244A - EGFP proteins with an anti - GFP antibody from nuclear extract of HEK293 cells . Immuno - blot detection was performed with antibodies recognizing GFP , histone variant H3 . 3 and CBX2 . ( L ) Percentage of Daxxm - z + zygotes with lagging chromosomes following injection of Tubulin - EGFP , Daxx - EGFP or DaxxR244A - EGFP mRNAs ( 20 or 100 ng / µl ) at PN2 / 3 stage . Data represent mean ± s . e . m . ( n ≥ 2 injection experiments ) . * * * P < 0 . 001 High Daxx - EGFP versus other 3 groups ; Fisher ' s exact test ( on pooled embryos ) . ( M ) Left : IF of EGFP and endogenous H3 at pat - PCH in Daxxm - z + PN5 zygotes expressing DAXX - EGFP or DAXXR244A - EGFP or nothing exogenously . Right : Fluorescence intensity profiles of H3 and EGFP along white lines in left panels at pat - PCH of zygotes . Scale bars 5 µm . Data information : All scale bars 10µm . ( N ) Boxplot displaying 3D quantification of H3 signal intensity at pat - PCH in Daxxm - z + zygotes expressing exogenous DAXX - EGFP ( n = 14 ) or DAXXR244A - EGFP ( n = 13 ) . In boxplots , the enter lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by R software ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 - 3 times . * * * P < 0 . 001 Daxx - EGFP versus other 2 groups ; t - test .

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

Figure 6(A) Developmental progression of Daxxm+z+, Daxxm-z+ and Daxxm-z- early embryos.(B) Examples of morula and blastocyst embryos with normal and large polyploid nuclei. Data information: All scale bars 10µm.(C) Percentages of embryos with normal or polyploid nuclei.(D) Still images of time-lapse imaging of the first cleavage division in wild-type and maternally deficient zygotes expressing H2B-mCherry. Time points represent minutes after prometaphase. Lagging chromosomes and micronuclei are indicated by yellow arrowheads. Data information: All scale bars 10µm.(E) Percentage of zygotes with lagging chromosomes during 1st cleavage division. Data represent mean ± s.e.m. in n≥3 replicate experiments.(F) IF of 5-methyl and 5-hydroxy-methyl cytosine staining of wild-type (n=42), Daxxm-z+ (n=14) and Ring1m-z+; Rnf2m-z+ (n=6) zygotes with lagging chromosomes. Yellow arrowheads indicate lagging chromosomes. Data information: All scale bars 10µm.(G) DNA-FISH for major (green) and minor (red) satellites on cleavage chromosomes of a Daxxm-z+ zygote with a chromosome broken within the major satellite region. Data information: All scale bars 10µm.(H) H3.3-EGFP live-imaging signal in pat- and mat-PCH of control Daxxm+z+ (n=18), Daxxm-z+ (n=8) and Ring1m-z+; Rnf2m-z+ (n=12) zygotes. Scale bar 10µm.(I) Left: IF of endogenous H3 and ATRX at pat-PCH in control and Daxxm-z+ PN5 zygotes. Right: Fluorescence intensity profiles of H3 and ATRX along white line in left panel at pat-PCH of zygotes. Scale bars 5 µm. Data information: All scale bars 10µm.(J) Boxplot displaying H3 signal intensity at pat-PCH of Daxxm+z+ and Daxxm-z+ PN5 zygotes. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test, n=12 for both groups.(K) IP of DAXX-EGFP and DAXXR244A-EGFP proteins with an anti-GFP antibody from nuclear extract of HEK293 cells. Immuno-blot detection was performed with antibodies recognizing GFP, histone variant H3.3 and CBX2.(L) Percentage of Daxxm-z+ zygotes with lagging chromosomes following injection of Tubulin-EGFP, Daxx-EGFP or DaxxR244A-EGFP mRNAs (20 or 100 ng/µl) at PN2/3 stage. Data represent mean ± s.e.m. (n ≥2 injection experiments). *** P<0.001 High Daxx-EGFP versus other 3 groups; Fisher's exact test (on pooled embryos).(M) Left: IF of EGFP and endogenous H3 at pat-PCH in Daxxm-z+ PN5 zygotes expressing DAXX-EGFP or DAXXR244A-EGFP or nothing exogenously. Right: Fluorescence intensity profiles of H3 and EGFP along white lines in left panels at pat-PCH of zygotes. Scale bars 5 µm. Data information: All scale bars 10µm.(N) Boxplot displaying 3D quantification of H3 signal intensity at pat-PCH in Daxxm-z+ zygotes expressing exogenous DAXX-EGFP (n=14) or DAXXR244A-EGFP (n=13). In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2-3 times. ***P<0.001 Daxx-EGFP versus other 2 groups; t-test.


Figure 7 ( A ) RNA FISH of forward and reverse strand major satellite sequences in Daxxm + z + and Daxxm - z + PN5 zygotes . Scale bars , 10µm . ( B ) Box plots indicating the foci number ( left ) and total intensity ( right ) of forward ( top ) and reverse ( bottom ) transcription as measured by RNA FISH on 3D paternal pronuclei in Daxxm + z + and Daxxm - z + PN5 zygotes . Lower hinge , central line and upper hinge in boxplots represent 25th , 50th ( median ) and 75th percentiles respectively . Upper / lower whiskers extend to the largest / smallest values no further than 1 . 5 * IQR from the upper / lower hinge , where IQR is inter - quartile range or distance between 25th and 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 3 times . * * * P < 0 . 001 ; t - test . ( C , D ) MA - plots showing Fold Change ( FC ) ( in log2 ) in gene expression between indicated samples as a function of averaged expression across all samples . Dotted lines indicated log2 ( FC ) of 1 . ( E ) Histogram showing classification of genes into 3 groups according to distribution of ChIP enrichment ( log2 ) of H3K27me3 around TSS in MII oocytes ( F ) MA - plot showing FC ( in log2 ) in gene expression between indicated samples as a function of averaged expression across all samples . Dotted lines indicated log2 ( FC ) of 1 . ( G - I ) Boxplots showing FC ( log2 ) in gene expression between indicated samples for 3 groups of genes classified according to H3K27me3 enrichments ( as shown in E ) . Lower hinge , central line and upper hinge represent 25th , 50th ( median ) and 75th percentiles respectively . Upper / lower whiskers extend to the largest / smallest values no further than 1 . 5 * IQR from the upper / lower hinge , where IQR is inter - quartile range or distance between 25th and 75th percentiles . Outliers are not displayed . The notches extend $ 1 . 5 * IQR / \ sqrt { n } $ where n is a number of genes in each H3K27me3 class . Number of genes ( n ) in " Low " , " Medium " and " High " H3K27me3 class used for boxplots are 9824 , 2311 , 2647 respectively . Expression changes were estimated using 2 or 3 biological replicates for each condition . Statistical significance was estimated using two - sided t - tests . ( J ) Boxplot showing FC ( log2 ) in gene expression between Daxx deficient and control MII oocytes for five groups of genes classified according to FC ( log2 ) in gene expression between Ring1 Rnf2 deficient and control GV oocytes . Boxplots were created and number of genes used for each boxplot is displayed below . Expression changes were estimated using 3 biological replicates for each condition . Statistical significance was estimated using two - sided t - tests .

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

Figure 7(A) RNA FISH of forward and reverse strand major satellite sequences in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Scale bars, 10µm.(B) Box plots indicating the foci number (left) and total intensity (right) of forward (top) and reverse (bottom) transcription as measured by RNA FISH on 3D paternal pronuclei in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Lower hinge, central line and upper hinge in boxplots represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001; t-test.(C, D) MA-plots showing Fold Change (FC) (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.(E) Histogram showing classification of genes into 3 groups according to distribution of ChIP enrichment (log2) of H3K27me3 around TSS in MII oocytes(F) MA-plot showing FC (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.(G - I) Boxplots showing FC (log2) in gene expression between indicated samples for 3 groups of genes classified according to H3K27me3 enrichments (as shown in E). Lower hinge, central line and upper hinge represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Outliers are not displayed. The notches extend $1.5*IQR/\sqrt{n}$ where n is a number of genes in each H3K27me3 class. Number of genes (n) in "Low", "Medium" and "High" H3K27me3 class used for boxplots are 9824, 2311, 2647 respectively. Expression changes were estimated using 2 or 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.(J) Boxplot showing FC (log2) in gene expression between Daxx deficient and control MII oocytes for five groups of genes classified according to FC (log2) in gene expression between Ring1 Rnf2 deficient and control GV oocytes. Boxplots were created and number of genes used for each boxplot is displayed below. Expression changes were estimated using 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.


Figure 1C - E Mapping of the binding region between myoVa and caldendrin using co - immunoprecipitation of cald - tagRFP and GFP - myoVa fragments expressed in HEK293T cells . Arrows in D indicate the size of the respective myosin fragment . F Summary of the results from C - E . Caldendrin binds specifically to a fragment containing IQ - motif 1 ( amino acids 742 - 791 ) , but not to any other region of myoVa , in a Ca2 + - dependent manner .

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

Figure 1C-E Mapping of the binding region between myoVa and caldendrin using co-immunoprecipitation of cald-tagRFP and GFP-myoVa fragments expressed in HEK293T cells. Arrows in D indicate the size of the respective myosin fragment. F Summary of the results from C - E. Caldendrin binds specifically to a fragment containing IQ-motif 1 (amino acids 742-791), but not to any other region of myoVa, in a Ca2+-dependent manner.


Figure 2B Western blot analysis of competition co - immunoprecipitation experiment in HEK293T cells . In the presence of Ca2 + , caldendrin binds strongly to the IQ1 fragment ( upper arrow ) , while tagRFP by itself does not bind ( lower arrow ) . Detection using a calmodulin antibody indicated that Ca2 + - dependent binding of caldendrin reduced the amount of CaM bound to the IQ1 motif relative to the control ( tagRFP only ) . MW = molecular weight marker . C Quantification of the normalized relative intensity of cald - tagRFP ( left ) and CaM bands ( right ) in western blot analysis of three independent experiments as shown in B . Shown is mean ± SEM . Upper : Ca2 + greatly increases cald - tagRFP binding to GFP - myoVa - IQ1 . Lower : When Ca2 + is available , the simultaneous presence of cald - tagRFP leads to a reduced binding of CaM compared to the control ( tagRFP ) . DF Native MS analysis of ion saturation for CaM . Ca2 + carried along from purification is detected on CaM in native MS . Without further supplementation with ions , intact CaM shows primarily its high affinity calcium binding sites occupied ( left ) , while in the same analysis myoVa peptide selectively and preferentially binds only to the fully Ca2 + - saturated CaM species ( right ) . Additional unassigned peaks represent unspecific adducts of Na + and Mg2 + ions . G Native MS analysis of ion saturation for caldendrin . Supplemented by an excess of 200 µM Ca2 + and 100 µM Mg2 + , caldendrin displays myoVa peptide binding to both fully Ca2 + - saturated and 2 Ca2 + - bound species ( right ) , which are also both detectable for the intact caldendrin protein in the same analysis ( left ) .

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 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 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

Figure 2B Western blot analysis of competition co-immunoprecipitation experiment in HEK293T cells. In the presence of Ca2+, caldendrin binds strongly to the IQ1 fragment (upper arrow), while tagRFP by itself does not bind (lower arrow). Detection using a calmodulin antibody indicated that Ca2+-dependent binding of caldendrin reduced the amount of CaM bound to the IQ1 motif relative to the control (tagRFP only). MW = molecular weight marker. C Quantification of the normalized relative intensity of cald-tagRFP (left) and CaM bands (right) in western blot analysis of three independent experiments as shown in B. Shown is mean ± SEM. Upper: Ca2+ greatly increases cald-tagRFP binding to GFP-myoVa-IQ1. Lower: When Ca2+ is available, the simultaneous presence of cald-tagRFP leads to a reduced binding of CaM compared to the control (tagRFP). DF Native MS analysis of ion saturation for CaM. Ca2+ carried along from purification is detected on CaM in native MS. Without further supplementation with ions, intact CaM shows primarily its high affinity calcium binding sites occupied (left), while in the same analysis myoVa peptide selectively and preferentially binds only to the fully Ca2+-saturated CaM species (right). Additional unassigned peaks represent unspecific adducts of Na+ and Mg2+ ions.G Native MS analysis of ion saturation for caldendrin. Supplemented by an excess of 200 µM Ca2+ and 100 µM Mg2+, caldendrin displays myoVa peptide binding to both fully Ca2+-saturated and 2 Ca2+-bound species (right), which are also both detectable for the intact caldendrin protein in the same analysis (left).


Figure 3C Visualization of F - actin gliding caused by myoVa activity . The first ( red ) and last ( green ) frame of a 30 second time - lapse image stack were color - coded and overlaid to visualize movement . Yellow = actin filament did not move during 30 seconds of imaging . In the presence of only CaM ( left ) , the majority of actin filaments are gliding . In the presence of both CaM and cald ( right ) , a large number of filaments are immobile . Scale bar = 10 µm .

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 3C Visualization of F-actin gliding caused by myoVa activity. The first (red) and last (green) frame of a 30 second time-lapse image stack were color-coded and overlaid to visualize movement. Yellow = actin filament did not move during 30 seconds of imaging. In the presence of only CaM (left), the majority of actin filaments are gliding. In the presence of both CaM and cald (right), a large number of filaments are immobile. Scale bar = 10 µm.


Figure 4B Co - sedimentation assay of GFP - tagged myosin fragment lacking the globular tail domain ( GFP - myoVa - motor + CC ) with F - actin shows that the presence of Ca2 + - caldendrin does not prevent myoV from binding to actin filaments . GFP - myoVa - motor + CC , as visualized by an anti - GFP antibody via western blot , co - sediments with F - actin ( comassie blue stained SDS - PAGE ) , both in presence and absence of Ca2 + , regardless of the presence of caldendrin . D Left : Representative image of COS7 cell expressing the inducible dimerization system described in C . Red : Peroxisomes ( PEX - RFP - FKBP ) , Green : KIF17 - GFP - PEX13 and myoV - GFP - FRB . Scale bar = 10 µm . Middle and right : Individual tracks of kinesin - driven peroxisome movements reconstructed over 20 seconds ( yellow ) before ( middle ) and after ( right ) addition of rapalog indicate that the recruitment of myoV slows down the motility of peroxisomes . Blue outlines represent manually drawn cellular border and nucleus ( excluded from analysis ) . E Analysis of the mean squared displacement ( MSD ) of peroxisomes over time ( left ) suggests a linear increase for all conditions . Slope analysis for MSD curves for individual cells ( right ) indicates that addition of rapalog induces efficient stalling ( 2 - way ANOVA , * * * p < 0 . 0001 as compared before and after rapalog ) . However , no difference between control and caldendrin ( cald ) transfected cells could be observed ( 2 - way ANOVA , Mean ± SEM ; n control = 14 cells , n cald = 23 cells ; 3 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 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 O O O O O

Figure 4B Co-sedimentation assay of GFP-tagged myosin fragment lacking the globular tail domain (GFP-myoVa-motor+CC) with F-actin shows that the presence of Ca2+-caldendrin does not prevent myoV from binding to actin filaments. GFP-myoVa-motor+CC, as visualized by an anti-GFP antibody via western blot, co-sediments with F-actin (comassie blue stained SDS-PAGE), both in presence and absence of Ca2+, regardless of the presence of caldendrin.D Left: Representative image of COS7 cell expressing the inducible dimerization system described in C. Red: Peroxisomes (PEX-RFP-FKBP), Green: KIF17-GFP-PEX13 and myoV-GFP-FRB. Scale bar = 10 µm. Middle and right: Individual tracks of kinesin-driven peroxisome movements reconstructed over 20 seconds (yellow) before (middle) and after (right) addition of rapalog indicate that the recruitment of myoV slows down the motility of peroxisomes. Blue outlines represent manually drawn cellular border and nucleus (excluded from analysis).E Analysis of the mean squared displacement (MSD) of peroxisomes over time (left) suggests a linear increase for all conditions. Slope analysis for MSD curves for individual cells (right) indicates that addition of rapalog induces efficient stalling (2-way ANOVA, *** p<0.0001 as compared before and after rapalog). However, no difference between control and caldendrin (cald) transfected cells could be observed (2-way ANOVA, Mean ± SEM; n control=14 cells, n cald=23 cells; 3 experiments).


Figure 5A Example confocal images from 10 min time - lapse imaging of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat primary hippocampal neurons ( DIV17 ) expressing an ER - marker ( ER - tDimer ) . Yellow arrows show dendritic protrusions with a stably inserted ER ( > 10 min ) . Blue arrows indicate protrusions with a transient ER entry . Scale bar = 5 µm . B Quantification of dynamic SER presence in protrusions of control ( GFP ) or caldendrin ( cald - GFP ) transfected neurons . In protrusions of cald - GFP neurons , the percentage of stably localized ER ( > 10 min ) is increased , while transient SER visits are decreased compared to the control . ( mean ± SEM , 2 - way ANOVA , * p = 0 . 0386 for interaction , n GFP = 9 , n cald - GFP = 10 ; 2 experiments ) C Representative confocal images of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat primary hippocampal neurons expressing an ER - marker ( ER - tDimer ) . To pre - load overexpressed caldendrin with Ca2 + , neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and stained with a homer1 - antibody to visualize synapse - containing spines . Arrows indicate spines that contain ER . Scale bar = 5 µm . D - F Quantification of protrusion - and spine density and the number of SER - positive spines as shown in C . ( mean ± SEM , n GFP ctrl = 24 cells , n Cald - GFP = 25 cells ; 3 experiments ) . ( D ) The density of dendritic protrusions ( spines and filopodia ) is increased in cald - GFP overexpressing neurons compared to the control . ( * p = 0 . 0119 , unpaired t - test ) . ( E ) The density of homer1 - positive dendritic spines is increased in cald - GFP overexpressing neurons compared to the control . ( * * p = 0 . 0049 , unpaired t - test ) . ( F ) The percentage of ER - containing dendritic spines is increased in cald - GFP overexpressing neurons compared to the control . ( * p = 0 . 0179 , unpaired t - test ) GG Representative confocal images of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat primary hippocampal neurons pre - treated with bicuculline as in C and stained against homer1 and the spine apparatus - marker synaptopodin . Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft , blue arrows indicate spine - localized synaptopodin . Scale bar = 5 µm . H - J Quantification of spine density , synaptopodin clusters and synaptopodin - positive spines as shown in G . ( mean ± SEM , n GFP ctrl = 21 cells , n cald - GFP = 30 cells ; 2 experiments ) . ( H ) Spine density is increased in cald - GFP overexpressing neurons compared to the control ( in line with 5e ) . ( * * p = 0 . 0024 , unpaired t - test ) . ( I ) The total number of synaptopodin clusters is not affected by caldendrin overexpression . ( n . s . , unpaired t - test ) . ( J ) The percentage of synapotpodin - positive spines is increased in cald - GFP overexpressing neurons compared to the control . ( * * * * p < 0 . 0001 , unpaired t - test ) .

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 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 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

Figure 5A Example confocal images from 10 min time-lapse imaging of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons (DIV17) expressing an ER-marker (ER-tDimer). Yellow arrows show dendritic protrusions with a stably inserted ER (>10 min). Blue arrows indicate protrusions with a transient ER entry. Scale bar = 5 µm. B Quantification of dynamic SER presence in protrusions of control (GFP) or caldendrin (cald-GFP) transfected neurons. In protrusions of cald-GFP neurons, the percentage of stably localized ER (> 10 min) is increased, while transient SER visits are decreased compared to the control. (mean ± SEM, 2-way ANOVA, * p=0.0386 for interaction, n GFP = 9, n cald-GFP = 10; 2 experiments)C Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons expressing an ER-marker (ER-tDimer). To pre-load overexpressed caldendrin with Ca2+, neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and stained with a homer1-antibody to visualize synapse-containing spines. Arrows indicate spines that contain ER. Scale bar = 5 µm. D - F Quantification of protrusion- and spine density and the number of SER-positive spines as shown in C. (mean ± SEM, n GFP ctrl = 24 cells, n Cald-GFP = 25 cells; 3 experiments). (D) The density of dendritic protrusions (spines and filopodia) is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0119, unpaired t-test). (E) The density of homer1-positive dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (** p=0.0049, unpaired t-test). (F) The percentage of ER-containing dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0179, unpaired t-test) GG Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons pre-treated with bicuculline as in C and stained against homer1 and the spine apparatus-marker synaptopodin. Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft, blue arrows indicate spine-localized synaptopodin. Scale bar = 5 µm. H - J Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in G. (mean ± SEM, n GFP ctrl = 21 cells, n cald-GFP = 30 cells; 2 experiments). (H) Spine density is increased in cald-GFP overexpressing neurons compared to the control (in line with 5e). (** p = 0.0024, unpaired t-test). (I) The total number of synaptopodin clusters is not affected by caldendrin overexpression. (n.s., unpaired t-test). (J) The percentage of synapotpodin-positive spines is increased in cald-GFP overexpressing neurons compared to the control. (**** p < 0.0001, unpaired t-test).


Figure 6A 10 min time - lapse imaging of DIV12 wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO + cald - GFP ) or caldendrin knock - out treated with jasplakinolide ( cald KO + JPK ) mouse primary hippocampal neurons , additionally expressing a cell fill ( maxGFP ) and an ER - marker ( ER - tDimer ) . Yellow arrows indicate protrusions with a stably anchored ER ( > 10 min ) . Blue arrows indicate protrusions that show transient presence of ER . Scale bar = 5 µm .

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

Figure 6A 10 min time-lapse imaging of DIV12 wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO + cald-GFP) or caldendrin knock-out treated with jasplakinolide (cald KO + JPK) mouse primary hippocampal neurons, additionally expressing a cell fill (maxGFP) and an ER-marker (ER-tDimer). Yellow arrows indicate protrusions with a stably anchored ER (>10 min). Blue arrows indicate protrusions that show transient presence of ER. Scale bar = 5 µm.


Figure 7A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin . The groups compared are wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO cald ) , and caldendrin knock - out treated with jasplakinolide ( cald KO JPK ) . Scale bar = 5 µm . B - C Quantification of spine density , synaptopodin clusters and synaptopodin - positive spines as shown in A ( mean ± SEM . Kruskal - Wallis test with Dunn ' s multiple comparisons post - test . * * * p < 0 . 0001 . cald wt n = 34 cells , 2 experiments ; cald KO n = 33 cells , 3 experiments ; cald KO cald n = 10 cells , 1 experiment ; cald KO JPK n = 14 cells , 1 experiment ) . ( B ) The total number of synaptopodin clusters is unaffected by the loss of caldendrin . ( C ) The number of synaptopodin - positive spines is reduced in cald KO compared to the WT neurons . This phenotype could be rescued by over - expression of caldendrin ( cald KO + cald ) , but not by treatment with JPK ( cald KO + JPK ) .

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 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

Figure 7A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin. The groups compared are wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO cald), and caldendrin knock-out treated with jasplakinolide (cald KO JPK). Scale bar = 5 µm.B - C Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in A (mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparisons post-test. *** p < 0.0001. cald wt n = 34 cells, 2 experiments; cald KO n = 33 cells, 3 experiments; cald KO cald n = 10 cells, 1 experiment; cald KO JPK n = 14 cells, 1 experiment). (B) The total number of synaptopodin clusters is unaffected by the loss of caldendrin. (C) The number of synaptopodin-positive spines is reduced in cald KO compared to the WT neurons. This phenotype could be rescued by over-expression of caldendrin (cald KO + cald), but not by treatment with JPK (cald KO + JPK).


Figure 1 The 3 ' - UTR sequence of mUcp1 RNA . Canonical CPEs ( UUUUA1 - 2U in bold ) , the reported BRF1 - binding site ( UAUUUAU , gray box ) and predicted IMP2 - binding motifs ( AYAYA and YA2YA , underlined ) are denoted . Ucp1L and Ucp1S carrying long and short 3 ' - UTRs , respectively , are resulted from alternative use of two polyadenylation signals ( Hex : AAUAAA , open box ) . ORF , open reading frame . The arrowhead marks the end of Ucp1S sequence . Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes Schematic comparison of mUcp1L and hUcp1 . Poly ( A ) signal ( Hex , open box ) and CPE ( black box ) are denoted . The RT - PCR results from human , WT ( Ucp1 + / + ) and UCP1 - KO ( Ucp1 - / - ) BAT RNA samples Dual luciferase reporter assay and RT - qPCR . The reporter plasmids , Fluc - mUcp1S , - mUcp1L or - hUcp1L 3 ' - UTR , and Rluc were co - transfected into differentiated HIB1B adipocytes . The transfected cells were used for luciferase activity assay and RT - qPCR . Data are mean ± SD from 3 independent experiments . Translation efficiency was defined as normalized activity ( Fluc / Rluc ) divided by normalized RNA level ( Fluc / Rluc ) . * * * p < 0 . 001 compared with Fluc - mUcp1S , one - way ANOVA

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 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

Figure 1 The 3'-UTR sequence of mUcp1 RNA. Canonical CPEs (UUUUA1-2U in bold), the reported BRF1-binding site (UAUUUAU, gray box) and predicted IMP2-binding motifs (AYAYA and YA2YA, underlined) are denoted. Ucp1L and Ucp1S carrying long and short 3'-UTRs, respectively, are resulted from alternative use of two polyadenylation signals (Hex: AAUAAA, open box). ORF, open reading frame. The arrowhead marks the end of Ucp1S sequence. Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes  Schematic comparison of mUcp1L and hUcp1. Poly(A) signal (Hex, open box) and CPE (black box) are denoted. The RT-PCR results from human, WT (Ucp1+/+) and UCP1-KO (Ucp1-/-) BAT RNA samples  Dual luciferase reporter assay and RT-qPCR. The reporter plasmids, Fluc-mUcp1S, -mUcp1L or -hUcp1L 3'-UTR, and Rluc were co-transfected into differentiated HIB1B adipocytes. The transfected cells were used for luciferase activity assay and RT-qPCR. Data are mean ± SD from 3 independent experiments. Translation efficiency was defined as normalized activity (Fluc/Rluc) divided by normalized RNA level (Fluc/Rluc). ***p < 0.001 compared with Fluc-mUcp1S, one-way ANOVA


Figure 2 Northern blotting of Ucp1 and 18S rRNA and RT - PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples ( 2 mice per genotype , 2 - 3 mo old ) . The short PCR fragments were isolated for sequencing Western blot analysis using WT and Ucp1 ∆ L BAT lysates . The protein level of UCP1 was normalized to GAPDH ( 4 mo old , n = 4 per group ) . * * p < 0 . 01 , Student ' s t test Dual luciferase reporter assay . The reporter plasmids , Fluc - mUcp1L 3 ' - UTR , and Rluc were co - transfected with the plasmid expressing EGFP , or myc - tagged CPEB ( immunoblots shown at the left ) into differentiated HIB1B adipocytes ( Data from 3 independent experiments ) . * p < 0 . 05 compared with EGFP , one - way ANOVA Western blot analysis using BAT lysates prepared from various CPEB - WT and KO male littermates ( 4 mo old , n = 4 per group ) . * p < 0 . 05 , Student ' s t test

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

Figure 2 Northern blotting of Ucp1 and 18S rRNA and RT-PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples (2 mice per genotype, 2-3 mo old). The short PCR fragments were isolated for sequencing  Western blot analysis using WT and Ucp1∆L BAT lysates. The protein level of UCP1 was normalized to GAPDH (4 mo old, n = 4 per group). **p < 0.01, Student's t test  Dual luciferase reporter assay. The reporter plasmids, Fluc-mUcp1L 3'-UTR, and Rluc were co-transfected with the plasmid expressing EGFP, or myc-tagged CPEB (immunoblots shown at the left) into differentiated HIB1B adipocytes (Data from 3 independent experiments). *p < 0.05 compared with EGFP, one-way ANOVA  Western blot analysis using BAT lysates prepared from various CPEB-WT and KO male littermates (4 mo old, n = 4 per group). *p < 0.05, Student's t test


Figure 3 Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2 - WT and - KO male mice at room temperature or after 5 - h cold exposure ( CE ) at 4oC ( 5 mice per genotype , 4 - 7 mo old )

O O O O O O O O O O O O O O O O O O O O O O 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 Northern blotting of Ucp1L and Ucp1S in BAT from CPEB2-WT and -KO male mice at room temperature or after 5-h cold exposure (CE) at 4oC (5 mice per genotype, 4-7 mo old)


Figure 4Western blot analysis and RT - qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT ( 13 mo old , n = 3 - 4 per group ) Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old ( n = 3 per group ) . Protein levels were normalized to that of GAPDH . RT - qPCR analysis of Ucp1 mRNA level in BAT collected from ( B ) relative to Gapdh mRNA levelHematoxylin and eosin sections of BAT from WT and CPEB2 - KO female mice at 4 and 12 mo old . Scale bars , 1 mmLipid droplet size and cell number ( i . e . , number of nuclei ) in WT and KO BAT ( representative images shown in 4D ) were quantified and displayed as arbitrary units . Three tissue sections from each BAT and 3 mice per group were analyzedWeight and representative images of WT and KO BAT . Three mice per group were

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

Figure 4Western blot analysis and RT-qPCR analysis of protein and mRNA levels of UCP1 and GAPDH in WT and KO male BAT (13 mo old, n = 3-4 per group)Western blot analysis of UCP1 and GAPDH in BAT lysates from WT and KO female mice at 4 and 12 mo old (n = 3 per group). Protein levels were normalized to that of GAPDH. RT-qPCR analysis of Ucp1 mRNA level in BAT collected from (B) relative to Gapdh mRNA levelHematoxylin and eosin sections of BAT from WT and CPEB2-KO female mice at 4 and 12 mo old. Scale bars, 1 mmLipid droplet size and cell number (i.e., number of nuclei) in WT and KO BAT (representative images shown in 4D) were quantified and displayed as arbitrary units. Three tissue sections from each BAT and 3 mice per group were analyzedWeight and representative images of WT and KO BAT. Three mice per group were analyzed


Figure 6 CPEB2 - WT and - KO mice were transduced with adeno - associated virus ( AAV ) expressing full - length ( AAV _ CP2 ) or RNA - binding domain ( AAV _ RBD ) of CPEB2 . Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV _ CP2 for 2 weeks , with Hoechst staining of nuclei . Scale bar , 10 µm . See more images in Fig EV4D Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction ( n = 6 mice per group ) . * p < 0 . 05 , Student ' s t test BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12 - h light time ( n = 7 mice per group ) . * p < 0 . 05 , Student ' s t test BAT temperature in WT and KO male mice , and of AAV _ CP2 - transduced WT and KO male mice ( n = 7 mice per group ) before and after i . p . injection of CL316243 ( CL ) . * p < 0 . 05 , repeated - measures two - way ANOVA

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 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

Figure 6 CPEB2-WT and -KO mice were transduced with adeno-associated virus (AAV) expressing full-length (AAV_CP2) or RNA-binding domain (AAV_RBD) of CPEB2. Immunostaining of CPEB2 and UCP1 in KO BAT transduced with AAV_CP2 for 2 weeks, with Hoechst staining of nuclei. Scale bar, 10 µm. See more images in Fig EV4D  Western blot analysis of UCP1 in BAT lysates from WT and KO male mice 2 weeks after AAV transduction (n = 6 mice per group). *p < 0.05, Student's t test  BAT temperature of WT and KO male mice after 2 weeks of AAV transduction measured during 12-h light time (n = 7 mice per group). *p < 0.05, Student's t test  BAT temperature in WT and KO male mice, and of AAV_CP2-transduced WT and KO male mice (n = 7 mice per group) before and after i.p. injection of CL316243 (CL). * p < 0.05, repeated-measures two-way ANOVA


Figure 1Micrographs collected in the GFP channel for each truncation ( columns ) of each studied MRP ( rows ) grouped by the N - terminus targeting properties based on theoretical expectation summarized in ( B ) with the MRPs possessing MTS - like N - termini in panel ( C ) Micrographs collected in the GFP channel for each truncation ( columns ) of each studied MRP ( rows ) grouped by the N - terminus targeting properties based on theoretical expectation summarized in ( B ) MRPs with intermediate phenotype in panel ( D ) and MRPs without N - terminal signal in panel ( E ) , for each MRP a yeast gene name and new nomenclature protein name is shown on the left . Scale bar for all micrographs is 5 µm .

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 O O O O O O O O O O O O O O O O O O O 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 1Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) with the MRPs possessing MTS-like N-termini in panel (C)Micrographs collected in the GFP channel for each truncation (columns) of each studied MRP (rows) grouped by the N-terminus targeting properties based on theoretical expectation summarized in (B) MRPs with intermediate phenotype in panel (D) and MRPs without N-terminal signal in panel (E), for each MRP a yeast gene name and new nomenclature protein name is shown on the left. Scale bar for all micrographs is 5 µm.


Figure 2 ( A ) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by fluorescent microscopy with MitoTracker Orange straining . Scale bar for all micrographs is 10 µm . ( B ) Characterization of Mrp17 translocation signal using an in vitro import assay : shown are autoradiographs of full - length Mrp17 or its truncations fused to DHFRmut , translated in vitro with radiolabeled amino - acids , incubated with isolated yeast mitochondria for 2 , 5 , or 10 min , treated with proteinase K ( PK ) to remove nonimported proteins and visualized by 16 % SDS - PAGE / autoradiography . As a negative control , mitochondria were treated with valinomycin , antimycin and oligomycin ( VAO ) that eliminate membrane potential . For comparison , 20 % of the protein used per import reaction was loaded on the first lane .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by fluorescent microscopy with MitoTracker Orange straining. Scale bar for all micrographs is 10 µm.(B) Characterization of Mrp17 translocation signal using an in vitro import assay: shown are autoradiographs of full-length Mrp17 or its truncations fused to DHFRmut, translated in vitro with radiolabeled amino-acids, incubated with isolated yeast mitochondria for 2, 5, or 10 min, treated with proteinase K (PK) to remove nonimported proteins and visualized by 16% SDS-PAGE/autoradiography. As a negative control, mitochondria were treated with valinomycin, antimycin and oligomycin (VAO) that eliminate membrane potential. For comparison, 20% of the protein used per import reaction was loaded on the first lane.


Figure 3 ( B ) In vitro mitochondria translocation capacity of WT Mrp17 , Mrp17K - R with all lysines ( K ) substituted with arginines ( R ) , and Mrp17K - A with all lysines ( K ) substituted with alanines ( A ) fused to DHFRmut . Import was performed as described in Fig . 2 legend . ( E ) Mrp17 variants rescue ∆ mrp17 strain growth defect on respiratory media : the indicated variants or empty vector ( EV ) were introduced in WT yeast and then the genomic MRP17 was disrupted by knock out , the resulting mutants were serially diluted 10 × and spotted on media containing glucose or glycerol as a sole carbon source .

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 O O O O O O O

Figure 3(B) In vitro mitochondria translocation capacity of WT Mrp17, Mrp17K-R with all lysines (K) substituted with arginines (R), and Mrp17K-A with all lysines (K) substituted with alanines (A) fused to DHFRmut. Import was performed as described in Fig. 2 legend.(E) Mrp17 variants rescue ∆mrp17 strain growth defect on respiratory media: the indicated variants or empty vector (EV) were introduced in WT yeast and then the genomic MRP17 was disrupted by knock out, the resulting mutants were serially diluted 10× and spotted on media containing glucose or glycerol as a sole carbon source.


Figure 4 ( A ) The in vitro import of Mrp17 - DHFRmut is sensitive to the elimination of outer mitochondrial membrane proteins by trypsinization ( left ) , more so than the model import substrate Atp1 ( right ) . Mitochondria were incubated with the indicated concentrations of trypsin and the import assay was performed as described in the legend for Fig . 2 . Proteinase K ( PK ) was not added to the sample without mitochondria . ( B ) In vitro translocation of Mrp17 - DHFRmut into mitochondria isolated from WT and ∆ tom20 yeast showing reduced translocation in ∆ tom20 background . ( G ) , ( H ) Import of Mrp17 - DHFRmut and control proteins Atp1 and Aac1 into WT , tim17 - ts ( G ) and tim22 - ts ( H ) mitochondria . Import was performed at indicated temperatures , proteinase K was added to all samples except the loading control in the first lane of each autoradiograph ( 20 % of protein amount used for each import reaction ) . The full autoradiograph for Aac1 with molecular weight markers is shown in Appendix Figure S6D .

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 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

Figure 4(A) The in vitro import of Mrp17-DHFRmut is sensitive to the elimination of outer mitochondrial membrane proteins by trypsinization (left), more so than the model import substrate Atp1 (right). Mitochondria were incubated with the indicated concentrations of trypsin and the import assay was performed as described in the legend for Fig. 2. Proteinase K (PK) was not added to the sample without mitochondria. (B) In vitro translocation of Mrp17-DHFRmut into mitochondria isolated from WT and ∆tom20 yeast showing reduced translocation in ∆tom20 background.(G),(H) Import of Mrp17-DHFRmut and control proteins Atp1 and Aac1 into WT, tim17-ts (G) and tim22-ts (H) mitochondria. Import was performed at indicated temperatures, proteinase K was added to all samples except the loading control in the first lane of each autoradiograph (20% of protein amount used for each import reaction). The full autoradiograph for Aac1 with molecular weight markers is shown in Appendix Figure S6D.


Figure 5 ( C ) Expression of structurally conserved bacterial RPs fused to GFP in yeast cells ( Full micrographs in al channels are shown on Fig . EV5 ) . Scale bar is 10 µm . ( E ) Chimeric variants of S6 depicted in panel D imported into isolated yeast mitochondria . Lanes are labeled as described in Fig . 2 legend .

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

Figure 5(C) Expression of structurally conserved bacterial RPs fused to GFP in yeast cells (Full micrographs in al channels are shown on Fig. EV5). Scale bar is 10 µm.(E) Chimeric variants of S6 depicted in panel D imported into isolated yeast mitochondria. Lanes are labeled as described in Fig. 2 legend.


Figure 6 ( B ) On the right : MRP N - termini structural environment ( solvent exposure ) : average number of atoms belonging to the same protein , other protein chains or RNA in the 13Å radius from the first structured amino acid Cα atom , plotted separately for proteins with their most N - terminus appearing in the structure vs proteins with precisely cleaved MTS , and other proteins which have a longer unstructured extension ( see the classification legend on the bottom ) calculated based on PDB : 5MRC ; on the right : same as on the left but only coordination number ( number of protein Cα atoms in the 13Å radius ( Hamelryck , 2005 ) ) is calculated , * * p - value < 0 . 01 ( Mann - Whitney test ) , absolute values were measured without replicates ; on the bottom : MRPs were divided in three groups depending on relative position of the MTS and structured residues : " No MTS ( str ) " - structure starts at the very N - terminus without a cleavable MTS , " Precise MTS " - MTS is cleaved off 1 - 2 amino acids before the structure starts , " Other " - unstructured uncleaved amino acids present before the structure start .

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

Figure 6(B) On the right: MRP N-termini structural environment (solvent exposure): average number of atoms belonging to the same protein, other protein chains or RNA in the 13Å radius from the first structured amino acid Cα atom, plotted separately for proteins with their most N-terminus appearing in the structure vs proteins with precisely cleaved MTS, and other proteins which have a longer unstructured extension (see the classification legend on the bottom) calculated based on PDB:5MRC; on the right: same as on the left but only coordination number (number of protein Cα atoms in the 13Å radius (Hamelryck, 2005)) is calculated, ** p-value<0.01 (Mann-Whitney test), absolute values were measured without replicates; on the bottom: MRPs were divided in three groups depending on relative position of the MTS and structured residues: "No MTS (str)" - structure starts at the very N-terminus without a cleavable MTS, "Precise MTS" - MTS is cleaved off 1-2 amino acids before the structure starts, "Other" - unstructured uncleaved amino acids present before the structure start.


Figure 1Histology : all metastatic lesions are composed of poorly differentiated cells with an epithelial ' flavour ' . The neoplastic population is mainly arranged in solid nests and sheets with focal rudimental gland formation . Scale bar : 50µm .

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

Figure 1Histology: all metastatic lesions are composed of poorly differentiated cells with an epithelial 'flavour'. The neoplastic population is mainly arranged in solid nests and sheets with focal rudimental gland formation. Scale bar: 50µm.


Figure 2Triplicate samples of L _ 03 , L _ 04A , L _ 07A , L _ 09 , L _ 10 ; L _ 12C are compared with the expression profiles deposited in the TCGA dataset of a spectrum of primary tumors or metastases ( meta ) from known origin . Two ovarian cancers analysed in house ( CTR _ OV1 and CTR _ OV2 ) were used as controls and have expression profiles matching the profiles displayed by the ovarian cancers listed in TCGA ( purple box ) . The acronyms are as follow : ACC : adrenocortical carcinoma ; BLCA : bladder urothelial carcinoma ; BRCA : breast invasive carcinoma ; CESC : cervical squamous cell carcinoma and endocervical adenocarcinoma ; CHOL : cholangiocarcinoma ; COAD : colon adenocarcinoma ; DLBC : lymphoid neoplasm diffuse large B - cell lymphoma ; ESCA : oesophageal carcinoma ; GBM : glioblastoma multiforme ; HNSC : head and neck squamous cell carcinoma ; KICH : kidney chromophobe ; KIRC : kidney renal clear cell carcinoma ; KIRP : kidney renal papillary cell carcinoma ; LAML : acute myeloid leukaemia ; LGG : brain lower grade glioma ; LIHC : liver hepatocellular carcinoma ; LUAD : lung adenocarcinoma ; LUSC : lung squamous cell carcinoma ; MESO : mesothelioma ; OV : ovarian serous cystadenocarcinoma ; PAAD : pancreatic adenocarcinoma ; PCPG : pheochromocytoma and paraganglioma ; PRAD : prostate adenocarcinoma ; READ : rectum adenocarcinoma ; SARC : sarcoma ; SKCM : skin cutaneous melanoma ; STAD : stomach adenocarcinoma ; TGCT : testicular germ cell tumors ; THCA : thyroid carcinoma ; THYM : thymoma ; UCEC : uterine corpus endometrial carcinoma ; UCS : uterine carcinosarcoma ; UVM : uveal melanoma .

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

Figure 2Triplicate samples of L_03, L_04A, L_07A, L_09, L_10; L_12C are compared with the expression profiles deposited in the TCGA dataset of a spectrum of primary tumors or metastases (meta) from known origin. Two ovarian cancers analysed in house (CTR_OV1 and CTR_OV2) were used as controls and have expression profiles matching the profiles displayed by the ovarian cancers listed in TCGA (purple box). The acronyms are as follow: ACC: adrenocortical carcinoma; BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; CESC: cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: cholangiocarcinoma; COAD: colon adenocarcinoma; DLBC: lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: oesophageal carcinoma; GBM: glioblastoma multiforme; HNSC: head and neck squamous cell carcinoma; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LAML: acute myeloid leukaemia; LGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma; MESO: mesothelioma; OV: ovarian serous cystadenocarcinoma; PAAD: pancreatic adenocarcinoma; PCPG: pheochromocytoma and paraganglioma; PRAD: prostate adenocarcinoma; READ: rectum adenocarcinoma; SARC: sarcoma; SKCM: skin cutaneous melanoma; STAD: stomach adenocarcinoma; TGCT: testicular germ cell tumors; THCA: thyroid carcinoma; THYM: thymoma; UCEC: uterine corpus endometrial carcinoma; UCS: uterine carcinosarcoma; UVM: uveal melanoma.


Figure 3Somatic mutations distribution : SNVs and InDels displayed by each metastasis ( listed in the Y axis ) were detected by Strelka2 tool ( Kim et al . , 2018 ) . Brown traits represent single mutations . The number of fully shared ( red ) , partially shared ( orange ) or private ( yellow ) mutations is indicated in the horizontal bar below . Five cancer - associated genes ( TP53 , ARID2 , NTRK1 , SMARCA4 and ZFHX3 ) mutated in all metastases are highlighted . Genetic similarity among metastases : the heatmap has been drawn according to the Jaccard index . The similarity ranges from 58 % to 82 % among all pairs with the exception of L _ 08 due to the scarce number of mutations . Variant allele frequency of fully shared mutations present in copy neutral regions . The box plot represents the fluctuation of the VAF ( in the Y axis ) of single mutations ( grey dots ) in each metastasis ; each box represents the upper and lower quartiles , while the central short black line within each box represents the median ; whiskers indicate variability outside the upper and lower quartiles .

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 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

Figure 3Somatic mutations distribution: SNVs and InDels displayed by each metastasis (listed in the Y axis) were detected by Strelka2 tool (Kim et al., 2018). Brown traits represent single mutations. The number of fully shared (red), partially shared (orange) or private (yellow) mutations is indicated in the horizontal bar below. Five cancer-associated genes (TP53, ARID2, NTRK1, SMARCA4 and ZFHX3) mutated in all metastases are highlighted.Genetic similarity among metastases: the heatmap has been drawn according to the Jaccard index. The similarity ranges from 58% to 82% among all pairs with the exception of L_08 due to the scarce number of mutations.Variant allele frequency of fully shared mutations present in copy neutral regions. The box plot represents the fluctuation of the VAF (in the Y axis) of single mutations (grey dots) in each metastasis; each box represents the upper and lower quartiles, while the central short black line within each box represents the median; whiskers indicate variability outside the upper and lower quartiles.


Figure 1A Coronal section of E15 . 5 wild - type ( WT ) and DKO developing cortices were co - stained with either anti - Sox2 ( green ) and anti - Pax6 ( red ) or with anti - Sox2 ( green ) and anti - Tbr2 ( red ) and both counter - stained with DAPI ( blue ) . Scale bars are 50 µm . Quantitation of total DAPI + cells and Sox2 + , Pax6 + or Tbr2 + progenitor cells in the SVZ / VZ in the entire image , corresponding to an area of 100 μM x 100 μM are plotted . B , D Coronal sections of WT and DKO developing cortices stained with DAPI ( blue ) and co - stained with anti - Ctip2 ( red ) and Satb2 ( green ) at P2 and P7 . The location of the cortical plate ( CP ) , intermediate zone ( IZ ) , subventricular / ventricular zone ( SVZ / VZ ) in P2 mice and Layers I - VI in P7 WT mice are indicated . Scale bars are 50 µm . C , E Quantitation of Ctip2 + and Satb2 + progenitor cells as a percent of total DAPI + cells in each of 8 bins ( marked on left ) within the cortex ( dashed lines ) from a total area corresponding to 100 μM x 600 μM are plotted .

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 O O O 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

Figure 1A Coronal section of E15.5 wild-type (WT) and DKO developing cortices were co-stained with either anti-Sox2 (green) and anti-Pax6 (red) or with anti-Sox2 (green) and anti-Tbr2 (red) and both counter-stained with DAPI (blue). Scale bars are 50 µm. Quantitation of total DAPI+ cells and Sox2+, Pax6+ or Tbr2+ progenitor cells in the SVZ/VZ in the entire image, corresponding to an area of 100 μM x 100 μM are plotted.B, D Coronal sections of WT and DKO developing cortices stained with DAPI (blue) and co-stained with anti-Ctip2 (red) and Satb2 (green) at P2 and P7. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in P2 mice and Layers I-VI in P7 WT mice are indicated. Scale bars are 50 µm.C, E Quantitation of Ctip2+ and Satb2+ progenitor cells as a percent of total DAPI+ cells in each of 8 bins (marked on left) within the cortex (dashed lines) from a total area corresponding to 100 μM x 600 μM are plotted.


Figure 2A - D Wild type ( WT ) , single ( Ltk - / - and Alk - / - ) and double ( DKO ) knockout embryos were injected with BrdU on day E14 . 5 and fixed cortices analyzed at E17 . 5 , P2 and P7 . A - C Coronal sections of BrdU labeled WT and mutant cortices at the indicated ages . Dotted lines mark the upper and lower cortical limits determined for each section . The location of the cortical plate ( CP ) , intermediate zone ( IZ ) , subventricular / ventricular zone ( SVZ / VZ ) in E17 . 5 and P2 mice and Layers I - VI in P7 WT mice are indicated . Scale bars are 50 µm . B , D Quantitation in E17 . 5 ( B ) or P2 and P7 ( D ) cortices of BrdU - positive cells in the indicated bins ( marked on left of images ) is plotted . Data are presented as the mean ± SEM from 3 ( DKO , Ltk - / - ) or 2 ( WT , Alk - / - ) independent biological experiments ( B ) or 3 ( DKO and WT ) independent biological experiments ( D ) . Statistical significance : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 005 , * p < 0 . 05 by Student ' s t - test .

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

Figure 2A-D Wild type (WT), single (Ltk-/- and Alk-/-) and double (DKO) knockout embryos were injected with BrdU on day E14.5 and fixed cortices analyzed at E17.5, P2 and P7. A-C Coronal sections of BrdU labeled WT and mutant cortices at the indicated ages. Dotted lines mark the upper and lower cortical limits determined for each section. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in E17.5 and P2 mice and Layers I-VI in P7 WT mice are indicated. Scale bars are 50 µm. B, D Quantitation in E17.5 (B) or P2 and P7 (D) cortices of BrdU-positive cells in the indicated bins (marked on left of images) is plotted. Data are presented as the mean ± SEM from 3 (DKO, Ltk-/-) or 2 (WT, Alk-/-) independent biological experiments (B) or 3 (DKO and WT) independent biological experiments (D). Statistical significance: ****p<0.0001, ***p<0.001, ** p<0.005, *p<0.05 by Student's t-test.


Figure 3B Dissociated E16 cortical neurons were cultured for 38 h and then fixed . Representative images of cortical neurons from WT , single or DKO mice , stained for Tau - 1 , an axonal marker ( red ) and MAP2 , a dendritic marker ( green ) . Arrowheads mark axons . Data are presented as percent of neurons with multiple , single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments . Statistical significance : * * * * p < 0 . 0001 , * * p < 0 . 01 , * p < 0 . 05 by one - way ANOVA , Dunnett ' s test . C Representative images of dissociated WT and DKO cortical neurons , electroporated with plasmids encoding EGFP alone or together with HA - tagged LTK - WT . Cells were fixed 38 h after plating and then stained for Tau - 1 ( axons , red ) and MAP2 ( dendrites , green ) . Arrowheads mark axons . Scale bars are 20 µm . Quantitation of the percent of neurons with multiple , single and no axons in EGFP positive neurons is plotted . Data are presented as percent of neurons with multiple , single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments . Statistical significance : * * * * p < 0 . 0001 , * * p < 0 . 01 , * p < 0 . 05 by one - way ANOVA , Dunnett ' s test . D Representative images of WT cortical neurons , treated with the ALK inhibitors , TAE684 and crizotinib or DMSO as a control at 4 h after plating . Neurons were stained for Tau - 1 ( red ) and MAP2 ( green ) . Arrowheads mark axons . : Data are presented as percent of neurons with multiple , single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments . Statistical significance : * * * * p < 0 . 0001 , * * p < 0 . 01 , * p < 0 . 05 by one - way ANOVA , Dunnett ' s test . E Golgi stains of adult cortices in wild - type , single ( Ltk - / - and Alk - / - ) and double ( DKO ) knockout mice show severe defects in neuronal projections and the callosal tract . Representative coronal sections of cortical layers I - V in WT and DKO mice ( left ) , Layers IV - V in WT , single and double knockout cortices ( center ) and higher magnification images of WT and DKO neurites ( right ) . Examples of disrupted directional growth and extension of projections in pyramidal neurons in knockout mice are marked ( red arrowheads ) . F Representative coronal sections of the medial region of the corpus callosum in wild - type and mutant cortices . Red line delineates the upper and lower regions of the corpus callosum ( CC ) with higher axon density within the upper corpus evident in WT mice . Prominent defects in the callosal fibers including substantial deviations and incoherent projections from the major tract in knockout mice compared to WT mice are marked ( red arrowheads ) . Schematics of the regions represented in images are shown on the right . CC : Corpus callosum , Py : pyramidal neurons .

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 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 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 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

Figure 3B Dissociated E16 cortical neurons were cultured for 38 h and then fixed. Representative images of cortical neurons from WT, single or DKO mice, stained for Tau-1, an axonal marker (red) and MAP2, a dendritic marker (green). Arrowheads mark axons. Data are presented as percent of neurons with multiple, single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test.C Representative images of dissociated WT and DKO cortical neurons, electroporated with plasmids encoding EGFP alone or together with HA-tagged LTK-WT. Cells were fixed 38 h after plating and then stained for Tau-1 (axons, red) and MAP2 (dendrites, green). Arrowheads mark axons. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single and no axons in EGFP positive neurons is plotted. Data are presented as percent of neurons with multiple, single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test.D Representative images of WT cortical neurons, treated with the ALK inhibitors, TAE684 and crizotinib or DMSO as a control at 4 h after plating. Neurons were stained for Tau-1 (red) and MAP2 (green). Arrowheads mark axons. : Data are presented as percent of neurons with multiple, single and no axons and are plotted as the mean ± SEM of 150 of neurons per condition from 3 independent biological experiments. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test.E Golgi stains of adult cortices in wild-type, single (Ltk-/- and Alk-/-) and double (DKO) knockout mice show severe defects in neuronal projections and the callosal tract. Representative coronal sections of cortical layers I-V in WT and DKO mice (left), Layers IV-V in WT, single and double knockout cortices (center) and higher magnification images of WT and DKO neurites (right). Examples of disrupted directional growth and extension of projections in pyramidal neurons in knockout mice are marked (red arrowheads).F Representative coronal sections of the medial region of the corpus callosum in wild-type and mutant cortices. Red line delineates the upper and lower regions of the corpus callosum (CC) with higher axon density within the upper corpus evident in WT mice. Prominent defects in the callosal fibers including substantial deviations and incoherent projections from the major tract in knockout mice compared to WT mice are marked (red arrowheads). Schematics of the regions represented in images are shown on the right. CC: Corpus callosum, Py: pyramidal neurons.


Figure 4C Schematic of the puzzle box arena consisting of a white , light compartment ( start box ) and a black compartment ( goal box ) divided by black barrier . Latency to reach the goal box during the 9 trials of the test is plotted for each individual 2 - 3 m old mouse of the indicated genotypes .

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

Figure 4C Schematic of the puzzle box arena consisting of a white, light compartment (start box) and a black compartment (goal box) divided by black barrier. Latency to reach the goal box during the 9 trials of the test is plotted for each individual 2-3 m old mouse of the indicated genotypes.


Figure 5Representative images of cortical neurons fixed at 38 h and stained with Tau - 1 ( axons , red ) and MAP2 ( dendrites , green ) are shown . Arrowheads mark axons . C , D Treatment of cortical neurons from WT or KO mice with PI3 kinase inhibitors reverses polarity defects caused by loss of Ltk / Alk . Dissociated E16 cortical neurons were treated with LY294002 ( 10 µM ; C ) , BKM120 ( 1 µM ; D ) or DMSO control , 4 h after plating . : In quantitation of the percent of neurons with multiple , single or no axons is plotted as the mean ± SEM of n = 150 neurons per condition from , C - WT ) or 4 ( C - DKO independent biological experiments . Statistical significance : * * * * E A representative immunoblotting showing the levels of pAKT ( pAktS473 ) , Akt and GAPDH ( loading control ) in lysates of cortical neurons from WT , single and DKO mice . Quantitation of pAktS473 to Akt levels relative to WT is plotted .

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 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

Figure 5Representative images of cortical neurons fixed at 38 h and stained with Tau-1 (axons, red) and MAP2 (dendrites, green) are shown. Arrowheads mark axons. C, D Treatment of cortical neurons from WT or KO mice with PI3 kinase inhibitors reverses polarity defects caused by loss of Ltk/Alk. Dissociated E16 cortical neurons were treated with LY294002 (10 µM; C), BKM120 (1 µM; D) or DMSO control, 4 h after plating. : In quantitation of the percent of neurons with multiple, single or no axons is plotted as the mean ± SEM of n=150 neurons per condition from , C-WT) or 4 (C-DKO independent biological experiments. Statistical significance: ****E A representative immunoblotting showing the levels of pAKT (pAktS473), Akt and GAPDH (loading control) in lysates of cortical neurons from WT, single and DKO mice. Quantitation of pAktS473 to Akt levels relative to WT is plotted.


Figure 6A , B IGF - 1R inhibitors rescue the multiple axon phenotype in DKO cortical neurons . Cortical neurons , isolated from WT and DKO mice were treated with 1 µM AG1024 ( A ) , varying concentrations of Picropodophylin ( PPP ; B ) or DMSO as control 4 h after plating , fixed at 38 h , and stained for Tau - 1 ( axons , red ) and MAP2 ( dendrites , green ) . Arrowheads mark axons . Scale bars are 20 µm . Quantitation of the percent of neurons with multiple , single or no axons is plotted . Data are presented as the mean ± SEM , n ≥ 150 neurons per condition from 4 independent biological experiments . C Blocking Igf - 1r activation with a neutralizing anti - IGF - 1Rα antibody rescued the multiple axon phenotype in DKO cortical neurons . Neurons isolated from WT or DKO mice were treated anti - IGF - 1Rα antibody ( 1 . 5 µg / ml ) , BSA as control ( 1 . 5 µg / ml ) , or PPP ( 200 nM ) 4 h after plating . Representative images of Tau - 1 and MAP2 stained cells with arrowheads marking axons are shown . Scale bars are 20 µm . Quantitation of the percent of neurons with multiple , single or no axons is plotted . F Inhibition of Igf - 1r blocks neuronal migration in vivo . Pregnant E14 WT or DKO mice were injected ( i . p ) with BrdU and 24 h later were injected with PPP or saline as control . Brains from P2 pups were subjected to staining with anti - BrdU antibody . Quantitation in P2 cortices of BrdU - positive cells in the indicated bins ( marked on left ) is plotted . The location of the cortical plate ( CP ) , intermediate zone ( IZ ) , subventricular / ventricular zone ( SVZ / VZ ) in P2 mice and are indicated . Scale bars are 50 µm . Data in ( F ) are presented as the mean ± SEM from 3 ( DKO ) and 2 ( WT ) independent biological experiments . Statistical significance : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 by one - way ANOVA , Dunnett ' s test by Student ' s t - test ( F ) , where WT vs WT + PPP or DKO vs DKO + PPP were compared .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B IGF-1R inhibitors rescue the multiple axon phenotype in DKO cortical neurons. Cortical neurons, isolated from WT and DKO mice were treated with 1 µM AG1024 (A), varying concentrations of Picropodophylin (PPP; B) or DMSO as control 4 h after plating, fixed at 38 h, and stained for Tau-1 (axons, red) and MAP2 (dendrites, green). Arrowheads mark axons. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single or no axons is plotted. Data are presented as the mean ± SEM, n ≥ 150 neurons per condition from 4 independent biological experiments.C Blocking Igf-1r activation with a neutralizing anti-IGF-1Rα antibody rescued the multiple axon phenotype in DKO cortical neurons. Neurons isolated from WT or DKO mice were treated anti-IGF-1Rα antibody (1.5 µg/ml), BSA as control (1.5 µg/ml), or PPP (200 nM) 4 h after plating. Representative images of Tau-1 and MAP2 stained cells with arrowheads marking axons are shown. Scale bars are 20 µm. Quantitation of the percent of neurons with multiple, single or no axons is plotted.F Inhibition of Igf-1r blocks neuronal migration in vivo. Pregnant E14 WT or DKO mice were injected (i.p) with BrdU and 24 h later were injected with PPP or saline as control. Brains from P2 pups were subjected to staining with anti-BrdU antibody. Quantitation in P2 cortices of BrdU-positive cells in the indicated bins (marked on left) is plotted. The location of the cortical plate (CP), intermediate zone (IZ), subventricular/ventricular zone (SVZ/VZ) in P2 mice and are indicated. Scale bars are 50 µm. Data in (F) are presented as the mean ± SEM from 3 (DKO) and 2 (WT) independent biological experiments. Statistical significance: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 by one-way ANOVA, Dunnett's test by Student's t-test (F), where WT vs WT+PPP or DKO vs DKO+PPP were compared.


Figure 7A Representative immunoblot showing the levels of phosphorylated ( pIgf - 1rY1135 / 1136 ) , total Igf - 1r and Gapdh ( as loading control ) in lysates of isolated cortical neurons from WT and DKO mice . Band intensities for Igf - 1r were normalized to Gapdh ( left plot ) and for pIgf - 1r were normalized either to Gapdh ( middle plot ) or to Igf - 1r ( right plot ) and the fold increase relative to WT is plotted . Data are presented as the mean ± SEM from 3 ( A independent experiments . Representative blots from 3 independent biological experiments are shown in ( AC Representative image showing phosphorylation of recombinant His - tagged IGF - 1R ( kinase domain and C - terminal region ) by recombinant GST - LTK ( kinase domain ) in an in vitro radioactive kinase assay . Phosphorylation of IGF - 1R was quantitated as fold change relative to auto - phosphorylation of IGF - 1R alone . Data in ( C are plotted as the mean ± SEM from 6 ( C ) independent biological experiments . E Endogenous ALK was immunoprecipitated from lysates of IMR - 32 cells , treated with control ( CCM ) or ALKAL2 ( ACM ) conditioned media and co - precipitated endogenous IGF - 1R was detected by immunoblotting . G Cell - surface expression of Igf - 1r is increased in DKO cortical neurons . Cell - surface Igf - 1r ( NA ppt ) is normalized to totals and the fold change relative to WT samples is plotted . I WT cortical neurons were labeled with biotin and incubated at 37ºC for 15 or 30 min in the presence of CCM or ACM conditioned media . Cell - surface Igf - 1r ( NA ppt ) normalized to totals as the fold change relative to controls at time 0 is plotted . J WT and DKO cortical neurons were treated with control ( CCM ) or ALKAL2 ( ACM ) conditioned medium 4 h after plating and IGF - 1 was added 1 hour later . Representative images of cells fixed at 36 h and stained for Tau - 1 ( axons ; red ) and MAP2 ( dendrites : green ) are shown . Arrowheads mark axons . Scale bars are 20 µm . In ( J ) quantitation of the percent of neurons with multiple , single or no axons is plotted as the mean ± SEM of n = 150 neurons per condition from 3 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7A Representative immunoblot showing the levels of phosphorylated (pIgf-1rY1135/1136), total Igf-1r and Gapdh (as loading control) in lysates of isolated cortical neurons from WT and DKO mice. Band intensities for Igf-1r were normalized to Gapdh (left plot) and for pIgf-1r were normalized either to Gapdh (middle plot) or to Igf-1r (right plot) and the fold increase relative to WT is plotted. Data are presented as the mean ± SEM from 3 (A independent experiments. Representative blots from 3 independent biological experiments are shown in (AC Representative image showing phosphorylation of recombinant His-tagged IGF-1R (kinase domain and C-terminal region) by recombinant GST-LTK (kinase domain) in an in vitro radioactive kinase assay. Phosphorylation of IGF-1R was quantitated as fold change relative to auto-phosphorylation of IGF-1R alone. Data in (C are plotted as the mean ± SEM from 6 (C) independent biological experiments.E Endogenous ALK was immunoprecipitated from lysates of IMR-32 cells, treated with control (CCM) or ALKAL2 (ACM) conditioned media and co-precipitated endogenous IGF-1R was detected by immunoblotting.G Cell-surface expression of Igf-1r is increased in DKO cortical neurons. Cell-surface Igf-1r (NA ppt) is normalized to totals and the fold change relative to WT samples is plotted.I WT cortical neurons were labeled with biotin and incubated at 37ºC for 15 or 30 min in the presence of CCM or ACM conditioned media. Cell-surface Igf-1r (NA ppt) normalized to totals as the fold change relative to controls at time 0 is plotted.J WT and DKO cortical neurons were treated with control (CCM) or ALKAL2 (ACM) conditioned medium 4 h after plating and IGF-1 was added 1 hour later. Representative images of cells fixed at 36 h and stained for Tau-1 (axons; red) and MAP2 (dendrites: green) are shown. Arrowheads mark axons. Scale bars are 20 µm. In (J) quantitation of the percent of neurons with multiple, single or no axons is plotted as the mean ± SEM of n=150 neurons per condition from 3 independent experiments.


Figure 1B Expression ( cells ) and secretion ( media ) of VgrG1 from V . parahaemolyticus RIMD 2210633 derivative POR1 ( Vpara ; T6SS1 + ) and its T6SS1 - mutant ( Vpara / ∆ hcp1 ) , and from V . natriegens ( Vnat ) carrying the indicated plasmids . Samples were treated ( + ) or not ( - ) with 20 µM phenamil to activate surface sensing in media containing 3 % NaCl at 30 ° C for 5 h . Loading control ( LC ) is shown for total protein lysates . Arrows denote bands corresponding to VgrG1 . Asterisks denote non - specific bands detected in Vnat samples . C Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 h ) co - incubation with the indicated attackers , as described in B , on media containing 3 % NaCl at 30 ° C . Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above . A significant difference was considered as P < 0 . 05 . DL , assay detection limit .

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 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

Figure 1B Expression (cells) and secretion (media) of VgrG1 from V. parahaemolyticus RIMD 2210633 derivative POR1 (Vpara; T6SS1+) and its T6SS1- mutant (Vpara/∆hcp1), and from V. natriegens (Vnat) carrying the indicated plasmids. Samples were treated (+) or not (-) with 20 µM phenamil to activate surface sensing in media containing 3% NaCl at 30 °C for 5 h. Loading control (LC) is shown for total protein lysates. Arrows denote bands corresponding to VgrG1. Asterisks denote non-specific bands detected in Vnat samples.C Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with the indicated attackers, as described in B, on media containing 3% NaCl at 30 °C. Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit.


Figure 2B Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 h ) co - incubation with wild type V . natriegens attackers harboring the indicated plasmids , on media containing 3 % NaCl at 30 ° C . Data information : data are shown as the mean ± SD , n = 3 technical replicates ; statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 . C Expression ( cells ) and secretion ( media ) of VgrG1 from V . natriegens containing an arabinose - inducible vp1407 in the chromosomal dns locus ( VnatReg ) and harboring an empty plasmid ( pEmpty ) or plasmids containing VpT6SS1 that lacks vp1407 ( pT6SS1Ind ) or both vp1407 and hcp1 ( pT6SS1Ind / ∆ hcp1 ; T6SS - ) . Samples were grown in media containing 3 % NaCl and either supplemented ( + ) or not ( - ) with 0 . 1 % arabinose ( Ara ) at 30 ° C . Loading control ( LC ) is shown for total protein lysates . E Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers carrying the indicated plasmids on solid media plates , as described in C . Data information : data are shown as the mean ± SD , n = 3 technical replicates ; statistical significance between samples at the 4 h timepoint by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as 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 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 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

Figure 2B Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with wild type V. natriegens attackers harboring the indicated plasmids, on media containing 3% NaCl at 30 °C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.C Expression (cells) and secretion (media) of VgrG1 from V. natriegens containing an arabinose-inducible vp1407 in the chromosomal dns locus (VnatReg) and harboring an empty plasmid (pEmpty) or plasmids containing VpT6SS1 that lacks vp1407 (pT6SS1Ind) or both vp1407 and hcp1 (pT6SS1Ind/∆hcp1; T6SS-). Samples were grown in media containing 3% NaCl and either supplemented (+) or not (-) with 0.1% arabinose (Ara) at 30 °C. Loading control (LC) is shown for total protein lysates.E Viability counts of V. natriegens prey before (0 h) and after (4 h) co-incubation with VnatReg attackers carrying the indicated plasmids on solid media plates, as described in C. Data information: data are shown as the mean ± SD, n = 3 technical replicates; statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.


Figure 3A , B Viability counts of V . natriegens prey before ( 0 h ) and after ( 4 or 24 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose ( A ) , or on media containing 0 . 1 % arabinose at 28 ° C ( B ) . Data information : Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 or 24 h timepoints by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as P < 0 . 05 . C - F Viability counts of the indicated prey bacteria before ( 0 h ) and after ( 4 h ) co - incubation with VnatReg attackers harboring the indicated plasmids , on media containing 3 % NaCl and 0 . 1 % arabinose at 28 ° C . Data information : Data are shown as the mean ± SD , n = 3 technical replicates . Statistical significance between samples at the 4 or 24 h timepoints by an unpaired , two - tailed Student ' s t - test is denoted above and is color coded to match the relevant samples . A significant difference was considered as 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B Viability counts of V. natriegens prey before (0 h) and after (4 or 24 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose (A), or on media containing 0.1% arabinose at 28 °C (B). Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.C-F Viability counts of the indicated prey bacteria before (0 h) and after (4 h) co-incubation with VnatReg attackers harboring the indicated plasmids, on media containing 3% NaCl and 0.1% arabinose at 28 °C. Data information: Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 or 24 h timepoints by an unpaired, two-tailed Student's t-test is denoted above and is color coded to match the relevant samples. A significant difference was considered as P < 0.05.