A Upper : representative showing that GS + MGs ( magenta ) were rarely observed in 5 - dpf jag2b - / - mutants expressing H2B - CFP ( green ) driven by crx promoter . Bottom : representative showing that GS + MGs ( magenta ) were frequently rescued underneath PRs when jag2b expression ( green ) was driven by crx promoter in 5 - dpf jag2b - / - mutants . of retina is labeled with solid , each layer structure is labeled with dashed lines marked with outer layer ( ONL ) , inner layer ( INL ) , inner plexiform layer ( IPL ) , ganglion layer ( GCL ) . Scale bars : 20μm . remaining MGs are indicated by arrowheads , rescued MGs are indicated by yellow arrows . B of GS + MGs ( magenta in A ) with ( green ) or without ( gray ) CFP expression driven by crx promoter . Ctrl indicates H2B - CFP expression driven by crx promoter ( n = 16 larvae ) ; rescue indicates jag2b - H2B - CFP expression driven by crx promoter ( n = 21 larvae , mean ± ; * p < 0 . 05 , ns , no significant difference , pair - wised t - ) . C proportions of jag2b - misexpressing PRs ( Green ) with underneath MGs ( GS + , magenta in A ) in 5 - dpf jag2b - / - mutants MGs in retina . In total , we analyzed retinae of 16 control ( Ctrl , jag2b - / - , crx : : h2b - ecfp expression ) 21 rescue ( jag2b - / - , crx : : jag2b expression ) larvae ( Mean ± ; * * * p < 0 . 001 , t - ) .

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A Upper: representative images showing that GS+ MGs (magenta) were rarely observed in 5-dpf jag2b-/- mutants expressing H2B-CFP (green) driven by the crx promoter. Bottom: representative images showing that GS+ MGs (magenta) were frequently rescued underneath PRs when jag2b expression (green) was driven by the crx promoter in 5-dpf jag2b-/- mutants. The area of retina is labeled with solid line, and each layer structure is labeled with dashed lines and marked with outer nuclear layer (ONL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Scale bars: 20μm. The remaining MGs are indicated by white arrowheads, and the rescued MGs are indicated by yellow arrows. B Quantification of GS+ MGs (magenta in A) with (green) or without (gray) CFP expression driven by the crx promoter. Ctrl indicates H2B-CFP expression driven by the crx promoter (n = 16 larvae); rescue indicates jag2b-H2B-CFP expression driven by the crx promoter (n = 21 larvae, mean ± SEM; * p<0.05, ns, no significant difference, pair-wised t-test). C The proportions of jag2b-misexpressing PRs (Green) with underneath MGs (GS+, magenta in A) in 5-dpf jag2b-/- mutants and all MGs in retina. In total, we analyzed retinae of 16 control (Ctrl, jag2b-/- , crx::h2b-ecfp expression) and 21 rescue (jag2b-/-, crx::jag2b expression) larvae (Mean ± SEM; ***p<0.001, t-test).


D Representative image showing GS + , but vsx1 - expression in progeny of Tp1 + Müller - like differentiating MGs at wild - type ( jag2b + / + ) . Layer structure indicated in ( A ) . E of progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expression GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in wild - type ( jag2b + / + ) fish ( n = 14 , Mean ± ; * * * p < 0 . 001 , t - ) . F Representative image showing vsx1 + , but GS - expression in progeny of Tp1 + Müller - like differentiating MGs in jag2b - / - mutants . Layer structure indicated in ( A ) . G of progeny of Müller - like Tp1 + differentiating MG precursors ( red + ) expressing GS ( gray ) , vsx1 ( green ) , or both negative ( magenta ) in jag2b - / - mutants ( n = 29 , mean ± ; * * * p < 0 . 001 , t - ) .

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D Representative image showing the GS+, but vsx1- expression in the progeny of Tp1+ Müller-like differentiating MGs at wild-type (jag2b+/+). Layer structure indicated in (A). E The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expression GS (gray), vsx1 (green), or both negative (magenta) in wild-type (jag2b+/+) fish (n = 14, Mean ± SEM; ***p<0.001, t-test). F Representative image showing vsx1+, but GS- expression in the progeny of Tp1+ Müller-like differentiating MGs in jag2b-/- mutants. Layer structure indicated in (A). G The percentage of the progeny of Müller-like Tp1+ differentiating MG precursors (red+) expressing GS (gray), vsx1 (green), or both negative (magenta) in jag2b-/- mutants (n = 29, mean ± SEM; ***p<0.001, t-test).


A Representative of Notch activation labeled by Tg ( Tp1 : mVenus ) ( green ) mature MGs stained with glutamate synthase ( GS , magenta ) at 4 dpf in Notch receptors ( notch1a - / - / notch1b - / - / notch3 - / - ) mutants . Scale Bars : 20 μm . B Quantitative plots showing numbers of with active Notch ( Tp1 + ) mature MG ( GS + ) at 4 dpf . In total , we collected 4 fish for wild - type fish ; 7 fish for notch1a - / - mutants ; 5 fish for notch1b - / - mutants ; 18 fish for notch3 - / - mutants ( Mean ± ; * * p < 0 . 01 ; * * * p < 0 . 001 , t - ) .

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A Representative images of Notch activation labeled by Tg(Tp1:mVenus)( green) and mature MGs stained with glutamate synthase (GS, magenta) at 4 dpf in Notch receptors (notch1a-/-/notch1b-/-/notch3-/-) mutants. Scale Bars: 20 μm. B Quantitative plots showing the numbers of cells with active Notch (Tp1+) and mature MG (GS+) cells at 4 dpf. In total, we collected 4 fish for wild-type fish; 7 fish for notch1a-/- mutants; 5 fish for notch1b-/- mutants; 18 fish for notch3-/- mutants (Mean ± SEM; ** p<0.01; ***p<0.001, t-test).


C Representative showing that GS + MGs ( magenta ) were rarely observed when H2B - CFP expression ( green ) was driven by gfap promoter in 5 - dpf jag2b - / - mutants . between upper dashed solid lines is outer layer ( ONL ) . in middle two dash lines is inner plexiform layer ( IPL ) . INL , inner layer ; GCL , ganglion layer . D Representative showing that GS + MGs ( magenta ) were frequently rescued underneath with notch3 expression ( green ) driven by gfap promoter in 5 - dpf jag2b - / - mutants . Layer structure is similar as ( C ) . E number of GS + MGs ( magenta ) with or without CFP expression driven by gfap promoter . Ctrl : H2B - CFP expression ( green ) driven by gfap promoter ( n = 5 fish ) ; rescue : notch1b ( n = 13 fish ) notch3 ( n = 17 fish ) NICD - H2B - CFP expression ( green ) driven by gfap promoter ( Mean ± ; * p < 0 . 05 , * * * p < 0 . 001 , ns , no significant difference , pair - wised t - ) . F proportions of notch1b notch3 NICD - misexpressing ( green ) with underneath MGs ( GS + , magenta ) in 5 - dpf jag2b - / - mutants , indicating that gfap promoter - driven notch1b ( n = 13 fish ) or notch3 ( n = 17 fish ) NICD expression could rescue MG loss in jag2b - / - mutants ( Mean ± ; * * * p < 0 . 001 , t - ) .

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C Representative images showing that GS+ MGs (magenta) were rarely observed when H2B-CFP expression (green) was driven by the gfap promoter in 5-dpf jag2b-/- mutants. The area between the upper dashed and solid lines is the outer nuclear layer (ONL). The area in the middle two dash lines is the inner plexiform layer (IPL). INL, inner nuclear layer; GCL, ganglion cell layer. D Representative images showing that GS+ MGs (magenta) were frequently rescued underneath cells with notch3 expression (green) driven by the gfap promoter in 5-dpf jag2b-/- mutants. Layer structure is similar as (C). E The cell number of GS+ MGs (magenta) with or without CFP expression driven by gfap promoter. Ctrl: H2B-CFP expression (green) driven by the gfap promoter (n = 5 fish); rescue: notch1b (n = 13 fish) and notch3 (n = 17 fish) NICD-H2B-CFP expression (green) driven by the gfap promoter (Mean ± SEM; * p<0.05, ***p<0.001, ns, no significant difference, pair-wised t-test). F The proportions of notch1b and notch3 NICD-misexpressing cells (green) with underneath MGs (GS+, magenta) in 5-dpf jag2b-/- mutants, indicating that gfap promoter-driven notch1b (n = 13 fish) or notch3 (n = 17 fish) NICD expression could rescue MG loss in jag2b-/- mutants (Mean ± SEM; ***p<0.001, t-test).


Maximum projection ( 2 z - slices ) of HeLa immunostained for F - actin ( Phalloidin ) , Tubulin , Pericentrin DAPI at metaphase early anaphase showing actin accumulation around centrosomes in early anaphase . Scale bar - 10µm of F - actin ( Phalloidin ) around pericentrin positive centrosomes in HeLa immunostained as in ( A ) , showing increase in F - actin around centrosomes at early anaphase . Mean actin metaphase = 1 ± 0 . 03996 , n = 92 , mean actin anaphase = 1 . 875 ± 0 . 08895 , n = 121 , Student t - , * * * * p < 0 . 0001 ;

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

Maximum projection (2 z-slices) of HeLa cells immunostained for F-actin (Phalloidin), Tubulin, Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase. Scale bar- 10µm Quantification of F-actin (Phalloidin) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in (A), showing the increase in F-actin around centrosomes at early anaphase. Mean actin metaphase = 1 ± 0.03996, n=92, mean actin anaphase = 1.875 ± 0.08895, n=121, Student t-test, ****p<0.0001;


Time - lapse from a representative Hela expressing Lifeact - GFP transiting from metaphase to anaphase , showing accumulation of actin in presumptive centrosomal region , red arrows . represent 4z - projection . Scale bar - 10µm .

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Time-lapse sequence from a representative Hela cell expressing Lifeact-GFP transiting from metaphase to anaphase, showing accumulation of actin in the presumptive centrosomal region, red arrows. Images represent 4z-projection. Scale bar-10µm.


Time - lapse of a representative HeLa expressing GFP - alpha - tubulin RFP - Lifeact transiting from metaphase to anaphase showing changes in actin microtubules . represent 4 z - projection of movies taken every 1min . Bottom - higher magnification view of centrosomes C1 C2 from time - lapse above showing a transient increase in actin around centrosomes in early anaphase . Scale bar = 10µm , 1µm in zoom . T = 0 is one frame before anaphase onset . Dotted circle shows centrosome position .

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Time-lapse sequence of a representative HeLa cell expressing GFP-alpha-tubulin and RFP-Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules. Images represent 4 z-projection of movies taken every 1min. Bottom - higher magnification view of the centrosomes C1 and C2 from time-lapse above showing a transient increase in actin around the centrosomes in early anaphase. Scale bar = 10µm, 1µm in zoom. T=0 is one frame before anaphase onset. Dotted circle shows centrosome position.


Integrated of GFP - alpha - tubulin RFP - Lifeact around centrosomes for a population of exiting mitosis , showing actin accumulation around centrosomes a simultaneous decrease in tubulin over time . N = 30 centrosomes ( 15 ) . T = 0 is one frame before anaphase onset . Graph shows mean + / - SD , values were normalized to at T = 0 . comparison of actin ( RFP - Lifeact ) tubulin ( a - tubulin - GFP ) around centrosomes at metaphase ( - 4 to - 1min ) , early anaphase ( 2 - 5min ) late anaphase ( 7 - 10min ) for a population of . Mean actin at Metaphase = 1 . 002 ± 0 . 0239 , n = 97 , Early Anaphase = 1 . 023 ± 0 . 03047 , n = 101 , Late anaphase = 0 . 9703 ± 0 . 05004 , n = 98 . Tubulin at Metaphase = 1 . 008 ± 0 . 03356 , n = 106 , Early anaphase = 0 . 9748 ± 0 . 04124 , n = 116 , Late anaphase = 0 . 9276 ± 0 . 05382 , n = 101 , One - way ANOVA .

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Integrated fluorescence intensity of GFP-alpha-tubulin and RFP-Lifeact around the centrosomes for a population of cells exiting mitosis, showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time. N=30 centrosomes (15 cells). T=0 is one frame before anaphase onset. Graph shows mean +/- SD, values were normalized to intensity at T=0. Quantification and comparison of actin (RFP-Lifeact) and tubulin (a-tubulin-GFP) fluorescence intensity around the centrosomes at metaphase (-4 to -1min), early anaphase (2-5min) and late anaphase (7-10min) for a population of cells. Mean actin intensity at Metaphase =1.002 ± 0.0239, n=97, Early Anaphase = 1.023 ± 0.03047, n=101, Late anaphase = 0.9703 ± 0.05004, n=98. Tubulin intensity at Metaphase = 1.008 ± 0.03356, n=106, Early anaphase = 0.9748 ± 0.04124, n=116, Late anaphase = 0.9276 ± 0.05382, n=101, One-way ANOVA.


Stills from time - lapse of HeLa expressing GFP - alpha - tubulin RFP - lifeact arrested at prometaphase with STLC ( t = 0 ) forced to exit mitosis with Cdk1 inhibition ( Ro3306 ) imaged every 90 seconds . Scale bar = 10µm for zoom = 4µm . n = 21 from 4 independent experiments .

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Stills from time-lapse of HeLa cells expressing GFP-alpha-tubulin and RFP-lifeact arrested at prometaphase with STLC (t=0) and forced to exit mitosis with Cdk1 inhibition (Ro3306) imaged every 90 seconds. Scale bar = 10µm and for zoom = 4µm. n = 21 cells from 4 independent experiments.


of actin around centrosomes when are forced to exit mitosis using Cdk1 inhibition as in ( A ) , showing a similar accumulation of actin as observed in bipolar divisions . Graph shows mean actin accumulation normalized to t = 0 , errors bars indicate standard deviation .

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Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in (A), showing a similar accumulation of actin as observed in bipolar divisions. Graph shows mean actin accumulation normalized to t=0, errors bars indicate standard deviation.


HeLa expressing GFP - centrin1 arrested at prometaphase or forced mitotic exit ( Ro3306 5 ' ) stained with phalloidin . Scale bar - 5µm for zoom - 2µm .

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HeLa cells expressing GFP-centrin1 arrested at prometaphase or forced mitotic exit (Ro3306 5') and stained with phalloidin. Scale bar- 5µm and for zoom-2µm.


level of actin around centrosome in ( C ) was quantified normalised relative to metaphase shows an increase during forced mitotic exit . STLC arrest 1 ± 0 . 01317 , n = 210 , STLC + Ro - 5mins = 1 . 251 ± 0 . 02441 , n = 144 , Unpaired t - with Welch correction , * * * * p < 0 . 0001 . of tubulin around centrosomes during prometaphase arrested or forced to exit with Cdk1 inhibitor . Tubulin decreses during forced mitotic exit . STLC - DMSO - 1 ± 0 . 02407 , n = 180 , STLC - RO - 3306 - 0 . 6516 ± 0 . 02337 , n = 136 . P < 0 . 0001 , Welch ' s t .

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The level of actin around the centrosome in (C) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit. STLC arrest 1 ± 0.01317, n=210, STLC+Ro-5mins=1.251 ± 0.02441, n=144, Unpaired t-test with Welch correction, ****p<0.0001. Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor. Tubulin intensity decreses during forced mitotic exit. STLC-DMSO- 1 ± 0.02407, n=180, STLC-RO-3306- 0.6516 ± 0.02337, n=136. P<0.0001, Welch's t test.


Maximum projection ( 2 - z slices ) view of Hela pre - treated with DMSO 0 . 2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around centrosomes during anaphase . Scale bar - 10µm . of actin around centrosomes for treated with DMSO or 0 . 2mM CK666 , showing reduction in actin accumulation around centrosomes following CK666 treatment . DMSO - metaphase - 1 ± 0 . 4325 , n = 54 , CK666 - metaphase - 0 . 936 ± 0 . 4604 , n = 43 , DMSO - anaphase - 1 . 8 ± 0 . 9736 , n = 76 , CK666 - anaphase - 1 . 087 ± 0 . 4597 , n = 73 ; one - way ANOVA , p < 0 . 0001 . Date pooled from 3 independent experiments .

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Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments.


Z - projection of HeLa expressing GFP - centrin1 pre - treated with DMSO or 0 . 2mM CK666 during prometaphase arrest forced mitotic exit stained with phalloidin for F - actin .

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Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin.


of level of actin around centrosome from ( C ) , which shows reduction in actin accumulation around centrosomes following CK666 pre - treatment . DMSO - STLC = 1 ± 0 . 02768 , n = 99 , CK666 STLC = 0 . 776 ± 0 . 02186 , n = 87 , DMSO - RO - 3306 = 1 . 339 ± 0 . 03048 , n = 127 , CK666 - RO - 3306 = 0 . 7699 ± 0 . 02246 , n = 70 , one - way ANOVA , p < 0 . 0001 .

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Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001.


of tubulin around centrosomes for treated with DMSO or 0 . 2mM CK666 , showing failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit . DMSO - metaphase - 1 ± 0 . 1968 , n = 54 , DMSO - anaphase - 0 . 867 ± 0 . 2345 , n = 78 , CK666 - metaphase - 0 . 7995 ± 0 . 1275 , n = 43 , CK666 - anaphase - 0 . 7407 ± 0 . 1859 , n = 74 ; one - way ANOVA , p < 0 . 0001 . Data pooled from 3 independent experiments . Error bars indicated standard deviation .

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Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation.


of tubulin around centrosomes for treated with DMSO or 0 . 2mM CK666 , during monopolar exit , showing failure to reduce tubulin density around centrosomes following CK666 treatment . Data from three independent experiments . Prometaphase arrest ( p . a . ) 1 ± 0 . 34 , n = 27 ; forced mitotic exit ( f . m . e ) 0 . 78 ± 0 . 30 , n = 26 ; p . a . plus CK666 0 . 49 ± 0 . 34 , n = 30 ; f . m . e plus CK666 0 . 92 ± 0 . 37 , n = 30 . One - way ANOVA with Sidak ' s multiple comparisons .

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Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.


Z - projection of HeLa expressing GFP - centrin1 immunostained for p34 - Arc ( sub - domain Arp2 / 3 ) following prometaphase arrest forced mitotic exit for 5min . N = 3 independent experiments . Scale bar = 10µm for zoom 2µm .

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Z-projection of HeLa cells expressing GFP-centrin1 immunostained for p34-Arc (sub-domain Arp2/3) following prometaphase arrest and forced mitotic exit for 5min. N=3 independent experiments. Scale bar = 10µm and for zoom 2µm.


of p34 - Arc around centrosome ( 3um ) were quantified relative to metaphase increase during forced mitotic exit . STLC arrested = 1 ± 0 . 01576 , n = 153 , STLC + RO - 3306 = 1 . 268 ± 0 . 01748 , n = 191 , * * * * p < 0 . 0001 , Unpaired t - with Welch correction . Error bars represent standard deviation .

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Levels of p34-Arc around the centrosome (3um area) were quantified relative to metaphase and increase during forced mitotic exit. STLC arrested = 1 ± 0.01576, n=153, STLC+RO-3306= 1.268 ± 0.01748, n=191, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.


Z - projection of centrosomes isolated from expressing GFP - centrin1 during prometaphase arrest forced exit , which were immunostained with p34 - Arc . N = 2 independent experiments . Scale bar = 2µm .

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Z-projection of centrosomes isolated from cells expressing GFP-centrin1 during prometaphase arrest and forced exit, which were immunostained with p34-Arc. N=2 independent experiments. Scale bar = 2µm.


of p34 - Arc around centrosome in ( C ) , relative to metaphase , showing its increase during forced exit . STLC arrested = 1 ± 0 . 00779 , n = 93 , STLC + RO - 3306 = 1 . 249 ± 0 . 02446 , n = 134 , * * * * p < 0 . 0001 , Unpaired t - with Welch correction . Error bars represent standard deviation .

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Quantification of p34-Arc levels around the centrosome in (C), relative to metaphase, showing its increase during forced exit. STLC arrested = 1 ± 0.00779, n=93, STLC+RO-3306= 1.249 ± 0.02446, n=134, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.


Time - lapse of in vitro on centrosomes isolated from prometaphase arrested forced to exit mitosis showing centrosomal actin nucleation over time . Scale bar = 10µm of actin nucleation from isolated centrosomes as in ( E ) over an of 2 μm , ( circle ) , showing an increase in actin nucleation around centrosomes isolated from undergoing forced mitotic exit . N = 2 isolations , 5 independent experiments , n = 16 STLC n = 19 STLC RO .

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Time-lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time. Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in (E) over an area of 2 μm, (white circle), showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit. N=2 isolations, 5 independent experiments, n=16 STLC and n=19 STLC RO.


Color switch experiment using green - red - labelled actin . Red actin was used first , followed by Capping , then green - labelled actin . accumulation of green in centre indicates that actin is nucleated at centrosome . Scale bar = 10µm . of ( G ) over time . N = 2 isolations , 2 independent experiments , n = 19 STLC n = 14 STLC RO . Error bars represent standard deviation .

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Color switch experiment using green- and red-labelled actin. Red actin was used first, followed by Capping protein, then green-labelled actin. The accumulation of green in the centre indicates that actin is nucleated at the centrosome. Scale bar = 10µm. Quantification of (G) over the time. N=2 isolations, 2 independent experiments, n=19 STLC and n=14 STLC RO. Error bars represent standard deviation.


Time - lapse showing actin filament formation at centrosomes isolated from undergoing forced exit when pre - treated with DMSO or CK666 . Scale bar = 10µm . of ( I ) , showing that centrosomal actin nucleation fails when centrosomes isolated from undergoing exit are pre - treated with CK666 . N = 2 isolations , 2 independent experiments , n = 17 DMSO , 26 Ro CK666 24 STLC DMSO , 23 STLC CK666 . Error bars represent standard deviation .

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Time-lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre-treated with DMSO or CK666. Scale bar = 10µm. Quantification of (I), showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre-treated with CK666. N=2 isolations, 2 independent experiments, n = 17 DMSO, 26 Ro CK666 24 STLC DMSO, 23 STLC CK666. Error bars represent standard deviation.


Phos - tag Western blot of MCF7 - Her2 - 18 synchronised into distinct phases of cycle showing phospho - shift in WASH1 during mitosis .

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Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis.


Blue native PAGE Western blot showing shift in of WASH1 isolated from STLC treated prometaphase arrested when compared to forced to exit mitosis following Ro3306 treatment .

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Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.


Time - lapse of HeLa expressing GFP - alpha tubulin RFP - lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase forced to exit mitosis with Ro3306 addition ( t = 0 ) . Scale bar = 5µm .

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Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm.


of actin around centrosome in ( A ) , showing failure to accumulate actin around centrosome in siWASH . N = 4 experiments . Error bars represent standard deviation .

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Quantification of actin around the centrosome in (A), showing the failure to accumulate actin around the centrosome in siWASH. N = 4 experiments. Error bars represent standard deviation.


Western blots of lysates from treated with siRNA control siRNA against WASH1 probed with WASH1 antibody , showing reduction in WASH following treatment of with siRNA against WASH . Scale bar = 5µm 2µm in zoom .

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Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody, showing the reduction in WASH levels following treatment of cells with siRNA against WASH. Scale bar = 5µm and 2µm in zoom.


Z - projection of HeLa expressing GFP - centrin1 treated with control siRNA or siRNA against WASH , arrested at prometaphase or forced to exit mitosis immunostained with phalloidin . Scale bar - 5µm of actin around centrosome in ( F ) , showing failure to accumulated actin around centrosome during forced exit in treated with siRNA against WASH . siControl - STLC = 1 ± 0 . 0189 , n = 165 , siWASH - STLC = 0 . 9138 ± 0 . 0298 , n = 118 , siControl - STLC + RO - 3306 = 1 . 911 ± 0 . 06221 , n = 147 , siWASH - STLC + RO - 3306 = 1 . 103 ± 0 . 0301 , n = 140 . Error bars represent standard deviation . P < 0 . 0001 , one - way ANOVA .

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Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA.


Time - lapse from in vitro with centrosomes isolated from undergoing forced exit were pre - treated with either no antibody or anti - WASH1 antibody for 1 hour . Scale bar - 10µm of in vitro from ( F ) , showing reduction in actin nucleation around centrosomes when they are pre - treated with anti - WASH1 . Error bars represent standard deviation . 2 independent experiments .

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Time-lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre-treated with either no antibody or anti-WASH1 antibody for 1 hour. Scale bar- 10µm Quantification of in vitro assay from (F), showing the reduction in actin nucleation around centrosomes when they are pre-treated with anti-WASH1. Error bars represent standard deviation. 2 independent experiments.


A TRAF6 expression in differentiating CD4 + T . Naïve CD4 + T were obtained from wild type C57BL / 6 mice by FACS activated with anti - CD3 / CD28 ( 1µg , 2µg / ml ) for indicated times in presence of distinct Thelper lineage - directing cytokines or under neutral activation conditions ( Th0 ) . After total extraction cDNA conversion , RT - PCR determined of Traf6 mRNA in differentiating Th0 , Th1 , Th17 iTregs was determined .

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A TRAF6 expression in differentiating CD4+ T cells. Naïve CD4+ T cells were obtained from wild type C57BL/6 mice by FACS and activated with anti-CD3/CD28 (1µg, and 2µg/ml) for the indicated times in the presence of distinct Thelper lineage-directing cytokines or under neutral activation conditions (Th0). After total RNA extraction and cDNA conversion, RT-PCR determined of Traf6 mRNA in differentiating Th0, Th1, Th17 and iTregs was determined.


B TRAF6 mRNA expression by human Tregs non - Treg CD4 + T . Human Tregs ( CD3 + / CD4 + / CD8 - / CD25HIGH / CD127low / CD39 + ) non - Treg CD4 + T ( CD3 + / CD4 + / CD8 - / CD25 - ) were obtained from peripheral blood of healthy donors by FACS after Ficoll - Paque PLUS gradient centrifugation magnetic bead enrichment of CD4 + T . TRAF6 mRNA was measured by qRT - PCR .

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B TRAF6 mRNA expression by human Tregs and non-Treg CD4+ T cell. Human Tregs (CD3+/CD4+/CD8-/CD25HIGH/CD127low/CD39+) and non-Treg CD4+ T cells (CD3+/CD4+/CD8-/CD25-) were obtained from the peripheral blood of healthy donors by FACS after Ficoll-Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4+ T cells. TRAF6 mRNA was measured by qRT-PCR.


C , D Evidence of lymphoproliferative disease in Traf6fl / flFoxp3Cre + mice . ( C ) Spleens lymph nodes were recovered from Traf6fl / flFoxp3Cre + mice Traf6fl / fl at 8 of ( Scale bars : 5 mm ) . ( D ) cellularity of lymphoid tissues of Traf6fl / flFoxp3Cre + mice their Traf6fl / fl Foxp3Cre - was determined ( 8 mice / group ) .

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C, D Evidence of lymphoproliferative disease in Traf6fl/flFoxp3Cre+ mice. (C) Spleens and lymph nodes were recovered from Traf6fl/flFoxp3Cre+ mice and Traf6fl/fl littermates at 8 weeks of age (Scale bars: 5 mm). (D) The cellularity of the lymphoid tissues of Traf6fl/flFoxp3Cre+ mice and their Traf6fl/fl Foxp3Cre- littermates was determined (8 mice/group).


E , H Effect of Treg - specific TRAF6 - deficiency on baseline T activation . frequencies of effector ( CD44high / CD62Llow ) , memory ( CD44high / CD62Lhigh ) naïve ( CD44low / CD62Lhigh ) in CD4 + T compartments of Traf6fl / fl Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) .

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E, H Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD4+ T cell compartments of Traf6fl/fl and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).


F , I Effect of Treg - specific TRAF6 - deficiency on baseline T activation . frequencies of effector ( CD44high / CD62Llow ) , memory ( CD44high / CD62Lhigh ) naïve ( CD44low / CD62Lhigh ) in CD8 + T compartments of Traf6fl / fl Foxp3Cre - ( wild type ) Traf6fl / flFoxp3Cre + mice were determined by flow cytometry ( 5 mice / group ) .

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F, I Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD8+ T cell compartments of Traf6fl/fl Foxp3Cre- (wild type) and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).


G , J Impact of TRAF6 - expression on in vitro Treg differentiation . naïve CD4 + T were isolated from Traf6fl / flFoxp3Cre + Traf6fl / fl mice differentiated into iTregs . Conditions of sub - optimal TGFβ concentrations ( 0 . 5ng / ml , 0 . 05ng / ml ) were tested as well intracellular FOXP3 was measured after 4 days .

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G, J Impact of TRAF6-expression on in vitro Treg differentiation. naïve CD4+ T cells were isolated from Traf6fl/flFoxp3Cre+ and Traf6fl/fl mice and differentiated into iTregs. Conditions of sub-optimal TGFβ concentrations (0.5ng/ml, 0.05ng/ml) were tested as well and intracellular FOXP3 was measured after 4 days.


A In vivo suppressive function of WT Traf6fl / flFoxp3Cre + Tregs . Tregs from indicated mice ( CD45 . 2 + ) were isolated by FACS , as were naïve ( CD62Lhigh / CD25 - ) CD4 + responder T from congenically distinct ( CD45 . 1 + ) donor mice . Tregs Tresponders were mixed at a 1 : 5 ( 2x105 : 10x105 ) before injection into Rag2 - / - mice . 7 days later , spleens were harvested , relative frequencies absolute numbers of each transferred populations were determined by flow cytometry .

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A In vivo suppressive function of WT and Traf6fl/flFoxp3Cre+ Tregs. Tregs from the indicated mice (CD45.2+) were isolated by FACS, as were naïve (CD62Lhigh/CD25-) CD4+ responder T cells from congenically distinct (CD45.1+) donor mice. Tregs and Tresponders were mixed at a 1:5 (2x105:10x105) ratio before injection into Rag2-/- mice. 7 days later, spleens were harvested, and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry.


B Implanted B16 melanoma growth in Traf6fl / flFoxp3Cre + wild type ( Traf6fl / flFoxp3Cre - ) mice . 1x105 B16F10 were injected subcutaneously ( s . c . ) into shaved flanks of indicated mice ( n = 5 / group ) . volumes were monitored every 3 days .

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B Implanted B16 melanoma growth in Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. 1x105 B16F10 cells were injected subcutaneously (s.c.) into the shaved flanks of the indicated mice (n= 5/group). Tumor volumes were monitored every 3 days.


C , D Proinflammatory cytokine production in - bearing mice with without Treg - specific TRAF6 expression . suspensions of - draining lymph node - infiltrating leukocytes ( TILs ) were recovered after 21 days of growth . Ex vivo stimulation with PMA ionomycin in presence of Golgistop for 5 hour preceded intracellular for IFNγ IL - 17 flow cytometry .

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C, D Proinflammatory cytokine production in tumor-bearing mice with and without Treg-specific TRAF6 expression. Cell suspensions of the tumor-draining lymph node and tumor-infiltrating leukocytes (TILs) were recovered after 21 days of tumor growth. Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL-17 and flow cytometry analysis.


E , F FOXP3 expression by CD4 + T in bearing Traf6fl / flFoxp3Cre + wild type ( Traf6fl / flFoxp3Cre - ) mice . frequency of FOXP3 + in CD4 + of - draining lymph nodes ( dLN ) , peripheral , non - draining lymph nodes ( pLN ) , spleens , TILs were determined by flow cytometry .

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E, F FOXP3 expression by CD4+ T cells in tumor bearing Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. The frequency of FOXP3+ cells in the CD4+ cells of tumor-draining lymph nodes (dLN), peripheral, non-tumor draining lymph nodes (pLN), spleens, and TILs were determined by flow cytometry.


A Assessment of K63 - type ubiquitination of FOXP3 upon expression of wild type catalytically deficient TRAF6 mutants . 293T were transfected with given combinations of vectors encoding wild type TRAF6 , enzymatically deficient L74H or C70A TRAF6 mutants , HA - FOXP3 , FLAG - labeled ubiquitin molecules possessing a single lysine residue ( K63 ) that restrain possible ubiquitin monomer linkages on polyubiquitinated to K63 - type only . were lysed modified by K63 - ubiquitin chains were recovered from lysates by pulling down using a bead - immobilized K63 - specific TUBE reagent . presence of FOXP3 among these modified was determined by immunoblotting as were of FOXP3 TRAF6 in pre - IP whole lysate ( WCL ) .

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A Assessment of K63-type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants. 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6, the enzymatically deficient L74H or C70A TRAF6 mutants, HA-FOXP3, and FLAG-labeled ubiquitin molecules possessing a single lysine residue (K63) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63-type only. Cells were lysed and proteins modified by K63-ubiquitin chains were recovered from lysates by pulling down using a bead-immobilized K63-specific TUBE reagent. The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre-IP whole cell lysate (WCL).


B Co - immunoprecipitation ( co - IP ) of FOXP3 with TRAF6 its facilitation of K63 - linked ubiquitination . 293T expressing indicated constructs encoding HA - FOXP3 FLAG - TRAF6 were lysed incubated with bead immobilized anti - FLAG antibodies . FOXP3 molecules co - IPed in this manner were resolved by SDS PAGE detected by immunoblotting with anti - HA antibodies .

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B Co-immunoprecipitation (co-IP) of FOXP3 with TRAF6 and its facilitation of K63-linked ubiquitination. 293T cells expressing the indicated constructs encoding HA-FOXP3 and FLAG-TRAF6 were lysed and incubated with bead immobilized anti-FLAG antibodies. FOXP3 molecules co-IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti-HA antibodies.


C 293T were transfected with indicated combinations of expression constructs encoding HA - FOXP3 , Myc - TRAF6 , FLAG - tagged ubiquitin molecules - either wild type ( FLAG - Ub ) , or a variant with a K - to - R mutation at either lysine residue 48 ( K48R ) or 63 ( K63R ) responsible for preventing K48 - K63 - type polyubiquitination , respectively . .

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C　293T cells were transfected with the indicated combinations of expression constructs encoding HA-FOXP3, Myc-TRAF6, and FLAG-tagged ubiquitin molecules - either wild type (FLAG-Ub), or a variant with a K-to-R mutation at either lysine residue 48 (K48R) or 63 (K63R) responsible for preventing K48- and K63-type polyubiquitination, respectively. .


D Endogenous , reciprocal co - IP of FOXP3 TRAF6 in murine iTreg lysate . Naïve CD62Lhigh / CD25 - / CD4 + T were obtained by FACS activated with anti - CD3 / CD28 antibodies ( 1ug 2ug / ml , respectively ) in presence of IL - 2 TGFβ ( 100U / ml 5ng / ml , respectively ) for four days before lysis . Antibodies against TRAF6 ( left ) or FOXP3 ( right ) were used to pull down target their interaction partners , which were resolved under denaturing conditions visualized by immunoblotting .

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D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting.


E Degree of K63 ubiquitination in FOXP3 pools of Traf6fl / flFoxp3Cre + Traf6wt / wtFoxp3Cre + mice . CD4 + / YFP + from Traf6fl / flFoxp3Cre + ( " Traf6 - / - " ) or wild type Traf6wt / wtFoxp3Cre + mice ( " Traf6 + / + " ) were isolated by FACS , lysed either total FOXP3 ( right ) or K63 - ubiquitin - modified ( left ) were immunoprecipitated . of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti - K63 - ubiquitin FOXP3 , respectively , of total FOXP3 actin were also measured by immunoblot of whole lysate .

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E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl/flFoxp3Cre+ and Traf6wt/wtFoxp3Cre+mice. CD4+/YFP+ cells from Traf6fl/flFoxp3Cre+ ("Traf6-/-") or wild type Traf6wt/wtFoxp3Cre+mice ("Traf6+/+") were isolated by FACS, lysed and either total FOXP3 (right) or K63-ubiquitin-modified proteins (left) were immunoprecipitated. Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti-K63-ubiquitin and FOXP3, respectively, and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate.


A Immunoblot of TRAF6 - mediated ubiquitination of wild type , mutant FOXP3 molecules . 293T lines were transfected with a normal HA - tagged Foxp3 construct , another encoding a ubiquitination resistant mutant in which lysine residues were replaced by arginines ( 20R ) , or one of 20 single lysine - containing constructs with only indicated lysine residue available for modification . These lines also received expression vectors encoding TRAF6 ubiquitin molecules labeled with Myc FLAG tags , respectively . Negative controls did not receive TRAF6 or expressed FOXP3 alone . Labeled FOXP3 were pulled down from lysates ( anti - HA ) ubiquitinated species were visualized by immunoblotting for FLAG .

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A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG.


B Assessing impact of K262 mutation on FOXP3 ubiquitination by TRAF6 . 293T were transfected with combinations of expression constructs encoding Myc - TRAF6 , FLAG - ubiquitin , an HA - tagged FOXP3 molecule that was either normal ( HA - FOXP3 ) or possessed a K - to - R mutation at residue 262 ( K262R ) . FOXP3 were pulled down with anti - HA beads either ubiquitinated or total FOXP3 were detected by probing for FLAG HA , respectively .

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B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6. 293T cells were transfected with combinations of expression constructs encoding Myc-TRAF6, FLAG-ubiquitin, and an HA-tagged FOXP3 molecule that was either normal (HA-FOXP3) or possessed a K-to-R mutation at residue 262 (K262R). FOXP3 proteins were pulled down with anti-HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA, respectively.


A Impact of K63 - ubiquitination loss on FOXP3 ' s gene silencing capacity . Jurkat T transfected with a wild type Foxp3 expression vector , one encoding K262R mutant , or an empty ( control ) vector also received a duel luciferase reporter construct where luciferase expression was under control of Il2 promoter . After activation with PMA ionomycin for 8 hours , luciferase activity was assayed .

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A Impact of K63-ubiquitination loss on FOXP3's gene silencing capacity. Jurkat T cells transfected with a wild type Foxp3 expression vector, one encoding the K262R mutant, or an empty (control) vector also received a duel luciferase reporter construct where luciferase expression was under the control of the Il2 promoter. After activation with PMA and ionomycin for 8 hours, luciferase activity was assayed.


B IL - 2 production in presence of wild type K63 - ubiquitination resistant FOXP3 . Naïve CD4 + T isolated from Traf6fl / flCD4Cre + or WT mice were activated overnight by anti - CD3 / CD28 antibodies , then transduced by retroviruses carrying normal Foxp3 , K262R mutant , or an empty vector . Ires - GFP in retroviral vector serves as an internal control . GFP + were sorted out 48 hours post - transduction . sorted were cultured for one additional day . Culture supernatants were collected for measuring of IL - 2 by ELISA .

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B IL-2 production in the presence of wild type and K63-ubiquitination resistant FOXP3. Naïve CD4+ T cells isolated from Traf6fl/flCD4Cre+ or WT mice were activated overnight by anti-CD3/CD28 antibodies, then transduced by retroviruses carrying normal Foxp3, the K262R mutant, or an empty vector. Ires-GFP in the retroviral vector serves as an internal control. GFP+ cells were sorted out 48 hours post-transduction. The sorted cells were cultured for one additional day. Culture supernatants were collected for measuring the levels of IL-2 by ELISA.


C distribution of wild type K262R FOXP3 . Hela were transfected with expression vectors encoding either wild type FOXP3 or K262R mutant . relative overlap of FOXP3 signal DAPI - stained nuclei was observed by microscopy . ( blue : DAPI , green : FOXP3 , Scale bars : 50 μm ) .

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C Cellular distribution of wild type and K262R FOXP3. Hela cells were transfected with expression vectors encoding either wild type FOXP3 or the K262R mutant. The relative overlap of FOXP3 protein signal and DAPI-stained nuclei was observed by fluorescence microscopy. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).


D Immunostaining of FOXP3 in Traf6fl / flCD4Cre + wild type ( Traf6fl / flCD4Cre - ) derived suspensions of lymph node spleen . ( blue : DAPI , green : FOXP3 , Scale bars : 50 μm ) .

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D Immunostaining of FOXP3 in Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) derived suspensions of lymph node and spleen cells. (blue: DAPI, green: FOXP3, Scale bars: 50 μm).


E of FOXP3 distribution in murine Tregs . degree of FOXP3 - coIocalization in CD4 + / FOXP3 + isolated from lymph nodes spleens of Traf6fl / flCD4Cre + wild type ( Traf6fl / flCD4Cre - ) mice was determined by Imagestream , frequencies of with either a low or high probability of FOXP3 distribution are shown ( red : stain , green : FOXP3 , Scale bars : 10 um ) .

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E Quantification of FOXP3 distribution in murine Tregs. The degree of FOXP3-nuclear coIocalization in CD4+/FOXP3+ cells isolated from the lymph nodes and spleens of Traf6fl/flCD4Cre+ and wild type (Traf6fl/flCD4Cre-) mice was determined by Imagestream analysis, and the frequencies of cells with either a low or high probability of nuclear FOXP3 distribution are shown (red: nuclear stain, green:FOXP3, Scale bars: 10 um).


in vitro suppressive potency of wild type - K262R Foxp3 - expressing T . Naïve CD4 + T were purified from Thy1 . 1 + BALB / c mice subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262 . These engineered " Tregs " were then co - cultured with naïve responder CD4 + T stained with CFSE at indicated ratios activated with anti - CD3 / CD28 antibodies . Dilution of CFSE signal by responder populations ( i . e . proliferation ) was assessed by flow cytometry .

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The in vitro suppressive potency of wild type- and K262R Foxp3-expressing T cells. Naïve CD4+ T cells were purified from Thy1.1+ BALB/c mice and subjected to retroviral transduction to express either wild type FOXP3 or a K262R mutant resistant to ubiquitination that lysine residue 262. These engineered "Tregs" were then co-cultured with naïve responder CD4+ T cells stained with CFSE at the indicated ratios and activated with anti-CD3/CD28 antibodies. Dilution of CFSE signal by the responder populations (i.e. proliferation) was assessed by flow cytometry.


C In vivo suppression of colitis by wild type - K262R mutant - Foxp3 expressing T . 2x105 naïve CD4 + transductants from A were mixed with 1x106 colitogenic naïve CD4 + T from wild type mice transferred i . v . into Rag2 - / - mice . Rag2 - / - mice receiving no transfers , naïve CD4 + T alone , or purified wild type Tregs served as controls ( n = 6 / group ) . Recipient mouse weights were monitored weekly for each group .

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C In vivo suppression of colitis by wild type- and K262R mutant-Foxp3 expressing T cells. 2x105 naïve CD4+ transductants from A were mixed with 1x106 colitogenic naïve CD4+ T cells from wild type mice and transferred i.v. into Rag2-/- mice. Rag2-/- mice receiving no cell transfers, naïve CD4+ T cells alone, or purified wild type Tregs served as controls (n= 6/group). Recipient mouse body weights were monitored weekly for each group.


D , E After 8 , colons were excised , fixed in buffered formalin , processed for H E ( D ) histopathology scoring ( E ) . Scale bars shown in 1 mm ) .

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D, E After 8 weeks, colons were excised, fixed in buffered formalin, and processed for H and E staining (D) and histopathology scoring (E). Scale bars shown in 1 mm).


B Western blot of liver lysates ( 100 µg ) shows that intein - mediated trans - splicing efficiently reconstitutes full - eGFP . Single AAV : n = 5 ; AAV - intein : n = 5 . Arrows indicate both full - eGFP excised inteins . C of eGFP bands . Values are reported as mean ± . Each dot represents eGFP band from injected with either single AAV n = 5 or AAV intein n = 5 .

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B Western blot analysis of liver lysates (100 µg) shows that intein-mediated protein trans-splicing efficiently reconstitutes full-length eGFP. Single AAV: n=5; AAV-intein: n=5. Arrows indicate both full-length eGFP and excised inteins. C Quantification of eGFP protein bands. Values are reported as mean±SEM. Each dot represents the eGFP protein band quantification from animals injected with either single AAV n=5 or AAV intein n=5.


B Western blot of lysates of HEK293 72 hours post - transfection with various F8 variants . Neg , non - transfected .

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B Western blot analysis of lysates of HEK293 cells 72 hours post-transfection with the various F8 variants. Neg, non-transfected cells.


C Chromogenic of F8 activity in medium of transfected are reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± . Each dot within same group corresponds to a biological replicate : Neg n = 5 ; Wild - type n = 8 ; N6 n = 8 ; SQ n = 7 ; V3 n = 6 . Significant differences between groups were assessed using Kruskal - Wallis followed by posthoc : Nemenyi ' s - Pairs Rank Comparison . Kruskal - Wallis p value = 2 . 88e - 05 . * * indicates significant difference between N6 Wild - type groups : p value ≤ 0 . 01 . * indicates significant difference between V3 Wild - type groups : p value ≤ 0 . 05 .

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C Chromogenic assay of F8 activity in the medium of transfected cells are reported as International Units/deciliter (IU/dl). Data are presented as mean±SEM. Each dot within the same group corresponds to a biological replicate: Neg n=5; Wild-type n=8; N6 n=8; SQ n=7; V3 n=6. Significant differences between groups were assessed using The Kruskal-Wallis test followed by the posthoc analysis: Nemenyi's All-Pairs Rank Comparison Test. The Kruskal-Wallis test p value = 2.88e-05. **indicates the significant difference between the N6 and the Wild-type groups: p value ≤ 0.01.*indicates the significant difference between the V3 and the Wild-type groups: p value ≤ 0.05.


A Western blot ( WB ) of lysates of HEK293 72 hours post - transfection ( n = 3 ; biological replicates ) with either Npu inteins or Heterologous ( N - intein DnaB + C - intein DnaE ) split - inteins . I + II , N6 split - intein ; I + II ( Het . ) , heterologous split - intein ; I , 5 ' N6 coding ( CDS ) - N - DnaE ; I ( N - int B ) , 5 ' N6 CDS - N - DnaB . II , C - DnaE - 3 ' N6 CDS . Excised inteins ( ∼ 12kDa ) are present only in down part of blot when I + II ( Npu inteins ) are provided . Arrows indicate full - N6 , single halves excised inteins .

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A Western blot (WB) of protein lysates of HEK293 cells 72 hours post-transfection (n=3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5'N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5'N6 CDS-N-DnaB. II, C-DnaE-3'N6 CDS protein. Excised inteins (∼12kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins.


B WB of medium of transfected showing secreted ( n = 3 ; biological replicates ) . Arrows indicate full - N6 as well as single halves excised inteins .

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B WB of medium of the transfected cells showing the secreted proteins (n=3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins.


C Chromogenic performed on medium of transfected to detect F8 activity reported as International Units / deciliter ( IU / dl ) . Data are presented as mean ± . Each dot within each group represent a different biological replicate ( n ) : I + II n = 3 ; I + II ( Het . ) n = 3 ; I ( N - int E ) n = 3 ; I ( N - int B ) n = 3 ; II ( C - int E ) n = 3 ; Neg n = 4 . Significant differences between groups were assessed using Kruskal - Wallis rank sum Kruskal − Wallis , p value = 0 . 013 . * indicates significant difference between I + II I ( N - int E ) groups : p value ≤ 0 . 05 . * * * indicates significant difference between I + II Neg groups : p value ≤ 0 . 001 .

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C Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/deciliter (IU/dl). Data are presented as mean ±SEM. Each dot within each group represent a different biological replicate (n): I + II n=3; I+II (Het.) n=3; I (N-int E) n=3; I (N-int B) n=3; II (C-int E) n=3; Neg n=4. Significant differences between groups were assessed using Kruskal-Wallis rank sum test Kruskal−Wallis, p value = 0.013. *indicates the significant difference between the I+II and the I (N-int E) groups: p value ≤ 0.05. ***indicates the significant difference between the I+II and the Neg groups: p value ≤0.001.


A Western blot ( WB ) of lysates of HEK293 72 hpt with AAV - N6 split - intein plasmids with codon - optimised set . I + II , N6 split - intein ; I , 5 ' N6 CDS - N - DnaE ; II , C - DnaE - 3 ' N6 CDS . ( n = 3 ; biological replicates ) . Arrows indicate full - N6 , excised inteins both single halves . Codop : codon - optimised .

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A Western blot (WB) of protein lysates of HEK293 cells 72 hpt with the AAV-N6 split- intein plasmids and with the codon-optimised set. I + II, N6 split-intein proteins; I, 5' N6 CDS-N-DnaE protein; II, C-DnaE-3'N6 CDS protein. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves. Codop: codon-optimised.


B WB of medium from transfected showing increased secretion of Codop - N6 full - compared to non - codon - optimised . ( n = 3 ; biological replicates ) . Arrows indicate full - N6 , excised inteins both single halves .

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B WB of medium from the transfected cells showing increased secretion of the Codop-N6 full-length protein compared to the non-codon-optimised. (n=3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves.


C Chromogenic performed on medium from transfected to measure F8 activity reported as International Units / deciliter ( IU / dl ) ( n = 3 ; biological replicates ) . Data are presented as mean ± . Significant differences between groups were assessed using Kruskal - Wallis p value = 0 . 027 . * indicates significant difference between I + II ( N6 split - intein ) II ( C - DnaE - 3 ' N6 CDS ) groups : p value ≤ 0 . 05 . * * indicates significant difference between Codop I + II I ( 5 ' N6 CDS - N - DnaE ) codop groups : p value ≤ 0 . 01 . * * indicates significant difference between Codop I + II II ( C - DnaE - 3 ' N6 CDS ) codop groups : p value ≤ 0 . 01 .

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C Chromogenic assay performed on the medium from transfected cells to measure F8 activity levels reported as International Units/deciliter (IU/dl) (n=3; biological replicates).Data are presented as mean ±SEM. Significant differences between groups were assessed using Kruskal-Wallis test p value = 0.027. *indicates the significant difference between the I+II (N6 split-intein) and the II (C-DnaE-3'N6 CDS) groups: p value ≤ 0.05. **indicates the significant difference between the Codop I+II and the I (5' N6 CDS-N-DnaE) codop groups: p value ≤ 0.01. **indicates the significant difference between the Codop I+II and the II (C-DnaE-3'N6 CDS) codop groups: p value ≤ 0.01.


A Alkaline gel Southern blot of AAV . AAV was hybridized to a probe specific for HLP promoter . Neg , AAV treated with Dnase I ; CodopV3 , AAV - CodopV3 ; I , AAV - 5 ' N6 - N - intein ; II , AAV - C - intein - 3 ' N6 .

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A Alkaline gel Southern blot analysis of AAV DNA. AAV DNA was hybridized to a probe specific for the HLP promoter. Neg, AAV DNA treated with Dnase I; CodopV3, AAV-CodopV3; I, AAV-5'N6-N-intein; II, AAV-C-intein-3' N6.


B Chromogenic performed on plasma samples to detect F8 activity in AAV - treated mice compared to controls groups . Data are presented as mean ± . same were analysed at various time - points . Each dot within same group within different groups of treatment corresponds to a single . baseline includes ( n = 20 ) before treatment : CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . statistical difference between groups has been assessed at 4weeks post - injection ( wpi ) with One - way Anova p value = 0 . 056 ; at 8 wpi with Kruskal - Wallis p value = 0 . 68 ; at 12wpi with Kruskal - Wallis p value = 0 . 45 ; at 16wpi with Kruskal - Wallis p value = 0 . 58 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromogenic assay performed on plasma samples to detect F8 activity in AAV-treated mice compared to controls groups. Data are presented as mean ±SEM. The same animals were analysed at the various time-points. Each dot within the same group and within different groups of treatment corresponds to a single animal. The baseline includes all animals (n=20) before the treatment: CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. The statistical difference between groups has been assessed at 4weeks post-injection (wpi) with the One-way Anova p value = 0.056; at 8 wpi with the Kruskal-Wallis test p value = 0.68; at 12wpi with the Kruskal-Wallis test p value = 0.45; at 16wpi with the Kruskal-Wallis test p value = 0.58.


C Activated partial thromboplastin time ( aPTT ) performed on plasma samples both at baseline at last time - point of ( 16 wpi ) . Data are presented as mean ± . Each dot within each group of treatment corresponds to different : baseline includes before treatment n = 21 ; CodopV3 : n = 8 ; N6 intein : n = 5 ; CodopN6 intein : n = 5 . Significant differences between groups were assessed using Kruskal - Wallis . * * indicates significant difference between baseline N6 intein groups : p value ≤ 0 . 01 . * indicates significant difference between baseline CodopN6 intein groups : p value ≤ 0 . 05 .

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

C Activated partial thromboplastin time (aPTT) assay performed on plasma samples both at the baseline and at the last time-point of the analysis (16 wpi). Data are presented as mean ±SEM. Each dot within each group of treatment corresponds to different animals: The baseline includes all animals before the treatment n=21; CodopV3: n=8; N6 intein: n=5; CodopN6 intein: n=5. Significant differences between groups were assessed using the Kruskal-Wallis test. **indicates the significant difference between the baseline and the N6 intein groups: p value ≤ 0.01.*indicates the significant difference between the baseline and the CodopN6 intein groups: p value ≤ 0.05.


D Tail - clip reported as bleeding time in seconds . Significant differences between groups were assessed using One - way Anova p value is = 0 . 11 . Data are presented as mean ± . Each dot corresponds to a different . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n =

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

D Tail-clip assay reported as bleeding time in seconds. Significant differences between groups were assessed using One-way Anova test p value is = 0.11.Data are presented as mean±SEM. Each dot corresponds to a different animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=


E Haemoglobin content measured as Optical Density ( OD ) at 416 nm after tail - clip . Significant differences between groups were assessed using One - way Anova . Data are reported as mean ± . Each dot corresponds to a single . Knockout ( K . O . ) group : n = 4 ; CodopV3 : n = 4 ; N6 intein : n = 4 ; CodopN6 intein : n = 6 .

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

E Haemoglobin content measured as Optical Density (OD) at 416 nm after the tail-clip assay. Significant differences between groups were assessed using One-way Anova test. Data are reported as mean±SEM. Each dot corresponds to a single animal. Knockout (K.O.) group: n=4; CodopV3: n=4; N6 intein: n=4; CodopN6 intein: n=6.


F Western blot of liver lysates ( 100 µg ) from either CodopN6 intein - treated mice ( n = 6 ) or untreated haemophilic mice ( n = 2 ) . A lysate from HEK293 transfected with N6 full - plasmid was used as positive control ( 50 µg ) . Arrows point at N6 full - ( N6 ) , 5 ' half of N6 ( 5 ' N6 ) 3 ' half of N6 ( 3 ' N6 ) .

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

F Western blot analysis of liver lysates (100 µg) from either CodopN6 intein-treated mice (n=6) or untreated haemophilic mice (n=2). A lysate from HEK293 cells transfected with the N6 full-length plasmid was used as positive control (50 µg). Arrows point at N6 full-length protein (N6), the 5' half of N6 (5'N6) and 3' half of N6 (3'N6).


A amount of anti - F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units / milliliter ( AU / ml ) . Each numbered bar represents a single mouse . CodopV3 , AAV - CodopV3 injected group n = 8 ; N6 intein , AAV - N6 intein injected group n = 5 ; CodopN6 intein , AAV - CodopN6 intein injected group n = 5 . Significant differences were assessed as follow : Paired T - has been used for CodopV3 n = 5 : p value ≤ 0 . 001 ; Wilcoxon for CodopV3 n = 3 : p value ≤ 0 . 05 ; Paired T - has been used for N6 intein n = 5 : p value ≤ 0 . 05 ; Paired T - has been used for CodopN6 intein n = 5 p value ≤ 0 . 01 .

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

A The amount of anti-F8 antibodies analysed by indirect ELISA is reported in Arbitrary Units/milliliter (AU/ml). Each numbered bar represents a single mouse. CodopV3, AAV-CodopV3 injected group n=8; N6 intein, AAV-N6 intein injected group n=5; CodopN6 intein, AAV-CodopN6 intein injected group n=5. Significant differences were assessed as follow: Paired T-test has been used for CodopV3 n=5: p value ≤ 0.001; Wilcoxon test for CodopV3 n=3: p value ≤ 0.05; Paired T-test has been used for N6 intein n=5: p value ≤ 0.05; Paired T- test has been used for CodopN6 intein n=5 p value ≤ 0.01.


B Bethesda performed on plasma samples mice injected with AAV - CodopV3 ; on plasma of injected with AAV - N6 intein n = 3 plasma of injected with AAV - CodopN6 intein n = 2 ; plasma of a wild - type was used as a control . dotted indicates threshold above which anti F8 - antibodies are considered inhibitors .

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

B Bethesda assay performed on plasma samples mice injected with AAV-CodopV3; on the plasma of animals injected with AAV-N6 intein n=3 and the plasma of animals injected with AAV-CodopN6 intein n=2; plasma of a wild-type animal was used as a control. The dotted line indicates the threshold above which anti F8-antibodies are considered inhibitors.


C Pro - inflammatory cytokines followed over - time reported as of baseline ( time point before injection ) . Data are represented as mean ± . Five belonging to CodopV3 group were analysed . Significant differences were assessed using Kruskal − Wallis ; for IP - 10 : * indicates p value ≤ 0 . 05 between 0 4 wpi ; * indicates p value ≤ 0 . 05 between 0 8 wpi ; * indicates p value ≤ 0 . 05 between 0 16 wpi . ( n = 20 ) ; for eotaxin : * indicates p value ≤ 0 . 05 .

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

C Pro-inflammatory cytokines levels followed over-time and reported as percentage of the baseline levels (time point before the injection). Data are represented as mean±SEM. Five animals belonging to the CodopV3 group were analysed. Significant differences were assessed using the Kruskal−Wallis test; for IP-10: * indicates the p value ≤ 0.05 between 0 and 4 wpi; * indicates the p value ≤ 0.05 between 0 and 8 wpi; * indicates the p value ≤ 0.05 between 0 and 16 wpi. (n=20); for eotaxin: * indicates the p value ≤ 0.05.


A Representative of eyes lenses from 6 - 7 week old wildtype ( WT ) nervous system - specific YME1L knockout ( NYKO ) mice . Orange dashed lines mark eye . Scale bars 5 mm .

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

A Representative images of eyes and lenses from 6-7 week old wildtype (WT) and nervous system-specific YME1L knockout (NYKO) mice. Orange dashed lines mark eye morphology. Scale bars 5 mm.


Retinal sagittal cross - sections from 6 - 7 week old mice stained with hematoxylin eosin . NFL = Nerve fiber layer , IPL = inner plexiform layer , INL = inner layer , OPL = outer plexiform layer , ONL = outer layer , R & C = Rods cones . Scale bars 30

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

Retinal sagittal cross-sections from 6-7 week old mice stained with hematoxylin and eosin. NFL = Nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = Rods and cones. Scale bars 30 µ


of nuclei in OPL ( = 1000 µm2 ) from retina cross - sections of 6 - 7 week old WT ( n = 4 ) NYKO ( n = 4 ) mice . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

Quantification of nuclei in OPL (area = 1000 µm2) from retina cross-sections of 6-7 week old WT (n = 4) and NYKO (n = 4) mice. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


D Immunoblot of retinal lysates from 6 - 7 week old WT NYKO mice . GFAP was used as a marker for reactive astrogliosis SDHA as a loading control . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

D Immunoblot analysis of retinal lysates from 6-7 week old WT and NYKO mice. GFAP was used as a marker for reactive astrogliosis and SDHA as a loading control. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


E mRNA of proinflammatory cytokines NF - κB target genes from 6 - 7 week old retinas ( WT , n = 5 ; NYKO , n = 5 ) . Transcript were normalized to Hprt mRNA . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

E mRNA levels of proinflammatory cytokines and NF-κB target genes from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


mRNA of Fgf21 from 6 - 7 week old retinas ( WT , n = 5 ; NYKO , n = 5 ) . Transcript were normalized to Hprt mRNA . Data were analyzed using unpaired t - , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

mRNA levels of Fgf21 from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


G Transmission electron of optic nerves from 6 - 7 week old WT NYKO mice . Scale bars 2 µm .

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

G Transmission electron micrographs of optic nerves from 6-7 week old WT and NYKO mice. Scale bars 2 µm.


Mean of wild type ( WT , n = 15 ) , NYKO ( n = 12 ) heterozygous NYKO ( het NYKO , n = 7 ) mice from 4 - 32 . Unpaired t - was used for comparison of two groups , ordinary one - way ANOVA for comparison of three groups . *

Mean body weight of male wild type (WT, n = 15), NYKO (n = 12) and heterozygous NYKO (het NYKO, n = 7) mice from 4-32 weeks. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *


B composition by magnetic resonance ( NMR ) of 31 - 32 week old WT ( n = 12 ) NYKO ( n = 7 ) mice . Unpaired t - was used for comparison of two groups , ordinary one - way ANOVA for comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Body composition analysis by nuclear magnetic resonance (NMR) of 31-32 week old male WT (n = 12) and NYKO (n = 7) mice. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


C Whole - energy expenditure per kg lean mass of 26 week old WT ( n = 5 ) NYKO ( n = 5 ) mice after disease onset . Unpaired t - was used for comparison of two groups , ordinary one - way ANOVA for comparison of three groups .

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

C Whole-body energy expenditure per kg lean mass of 26 week old male WT (n = 5) and NYKO (n = 5) mice after disease onset. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups.


Walking beam of 6 - 7 week old ( WT , n = 10 ; NYKO , n = 10 ) , 17 - 18 week old ( WT , n = 8 ; NYKO , n = 9 ) 31 - 32 week old mice ( WT , n = 12 ; NYKO , n = 19 ; het NYKO , n = 9 ) . Unpaired t - was used for comparison of two groups , ordinary one - way ANOVA for comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± .

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

Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


Walking beam of 6 - 7 week old ( WT , n = 10 ; NYKO , n = 10 ) , 17 - 18 week old ( WT , n = 8 ; NYKO , n = 9 ) 31 - 32 week old mice ( WT , n = 12 ; NYKO , n = 19 ; het NYKO , n = 9 ) . Hind limb slips per run are shown in E . Unpaired t - was used for comparison of two groups , ordinary one - way ANOVA for comparison of three groups . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Data are means ± .

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

Walking beam test of 6-7 week old (WT, n = 10; NYKO, n = 10), 17-18 week old (WT, n = 8; NYKO, n = 9) and 31-32 week old mice (WT, n = 12; NYKO, n = 19; het NYKO, n = 9). Hind limb slips per run are shown in E. Unpaired t-test was used for the comparison of two groups, ordinary one-way ANOVA for the comparison of three groups. *P ≤ 0.05, *** P ≤ 0.001, ****P ≤ 0.0001. Data are means ± SEM.


F Representative of 31 - 32 week old WT NYKO mice showing aberrant positioning of hind limbs .

F Representative images of 31-32 week old WT and NYKO mice showing aberrant positioning of hind limbs.


Brain weights were monitored at 31 - 32 of ( WT , n = 15 ; NYKO , n = 14 ) . Unpaired t - , ns = not significant . Data are means ± .

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

Brain weights were monitored at 31-32 weeks of age (WT, n = 15; NYKO, n = 14). Unpaired t-test, ns = not significant. Data are means ± SEM.


B Nissl stainings of sagittal sections across brain regions from 31 - 32 week old WT NYKO mice . 4x 40x enlargements are shown in left right panels , respectively . Scale bars 4x = 500 µm , 40x = 50 µm .

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

B Nissl stainings of sagittal sections across brain regions from 31-32 week old WT and NYKO mice. 4x and 40x enlargements are shown in the left and right panels, respectively. Scale bars 4x = 500 µm, 40x = 50 µm.


C Morphometric of different brain regions from 31 - 32 week old WT ( n = 6 ) NYKO ( n = 6 ) mice . GCL = Granule layer , ML = Molecular layer . Unpaired t - , ns = not significant . Data are means ± .

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

C Morphometric analysis of different brain regions from 31-32 week old WT (n = 6) and NYKO (n = 6) mice. GCL = Granule cell layer, ML = Molecular layer. Unpaired t-test, ns = not significant. Data are means ± SEM.


Immunoblot of tissue lysates ( FB = forebrain ; CB = cerebellum ) from 31 - 32 week old WT NYKO mice , using GFAP - specific , as a loading control , GAPDH - specific antibodies . Unpaired t - , ns = not significant . Data are means ± .

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

Immunoblot analysis of tissue lysates (FB = forebrain; CB = cerebellum) from 31-32 week old WT and NYKO mice, using GFAP-specific and, as a loading control, GAPDH-specific antibodies. Unpaired t-test, ns = not significant. Data are means ± SEM.


B Transverse semithin sections of spinal cords of WT NYKO mice of indicated . Sections were stained with toluidine blue . Orange arrows indicate degenerating neurons ( Myelin dense bodies , MDBs ) . Scale bars 25 µm .

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

B Transverse semithin sections of spinal cords of WT and NYKO mice of the indicated age. Sections were stained with toluidine blue. Orange arrows indicate degenerating neurons (Myelin dense bodies, MDBs). Scale bars 25 µm.


C Myelin dense bodies ( MDBs ) in dorso - lateral tracts of 6 - 7 week old WT ( n = 4 ) NYKO ( n = 4 ) , 17 - 18 week old WT ( n = 4 ) NYKO ( n = 4 ) 31 - 32 week old WT ( n = 6 - 7 ) NYKO ( n = 5 - 6 ) mice spinal cords . Unpaired t - , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

C Myelin dense bodies (MDBs) in dorso-lateral tracts of 6-7 week old WT (n = 4) and NYKO (n = 4), 17-18 week old WT (n = 4) and NYKO (n = 4) and 31-32 week old WT (n = 6-7) and NYKO (n = 5-6) mice spinal cords. Unpaired t-test, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


D Immunoblots of 31 - 32 week old spinal cord lysates . GAPDH was used to control for gel loading .

D Immunoblots of 31-32 week old spinal cord lysates. GAPDH was used to control for gel loading.


E mRNA of proinflammatory cytokines from spinal cords of 31 - 32 week old mice ( WT , n = 5 ; NYKO , n = 5 ) . Transcript were normalized to Hprt mRNA . Unpaired t - , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± .

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

E mRNA levels of proinflammatory cytokines from spinal cords of 31-32 week old mice (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Unpaired t-test, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


of isolated cortical neurons from Yme1lfl / fl expressing CAG - GFP ( WT ) or CAG - Cre - IRES - GFP ( Yme1l - / - ) at DIV12 stained with TOMM20 to visualize mitochondria . Scale bars 30 µm .

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

Fluorescence images of isolated cortical neurons from Yme1lfl/fl animals expressing CAG-GFP (WT) or CAG-Cre-IRES-GFP (Yme1l-/-) at DIV12 and stained with TOMM20 to visualize mitochondria. Scale bars 30 µm.


B - E of total axon ( WT , n = 13 neurons ; Yme1l - / - , n = 13 neurons ) , total dendritic ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) , axonal branch points ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) number of dendrites ( WT , n = 12 neurons ; Yme1l - / - , n = 13 neurons ) . neurons originate from two independent preparations . Unpaired t - , ns = not significant , * P ≤ 0 . 05 .

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

B - E Quantification of total axon length (WT, n = 13 neurons; Yme1l-/-, n = 13 neurons), total dendritic length (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons), axonal branch points (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons) and number of dendrites (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons). Individual neurons originate from two independent preparations. Unpaired t-test, ns = not significant, *P ≤ 0.05.


F , G Kymographs showing mitochondrial movements in representative axons . Vertical lines correspond to stationary mitochondria diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 39 axonal segments were analyzed for WT control neurons 37 axonal segments for Yme1l - / - neurons at DIV7 . Data are means of three independent experiments ± . Unpaired t - , ns = not significant , * P ≤ 0 . 05 .

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

F, G Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 39 axonal segments were analyzed for WT control neurons and 37 axonal segments for Yme1l-/- neurons at DIV7. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


H , I Kymographs showing mitochondrial movements in representative axons . Vertical lines correspond to stationary mitochondria diagonal lines to moving mitochondria . Mitochondria were stained using Mitotracker . In total 120 axonal segments were analyzed for WT control neurons 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± . Unpaired t - , ns = not significant , * P ≤ 0 . 05 .

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

H, I Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.


J Axonal occupied by mitochondria in Yme1l - / - neurons relative to WT controls . In total 120 axonal segments were analyzed for WT control neurons 107 axonal segments for Yme1l - / - neurons at DIV10 - 14 . Data are means of three independent experiments ± . Unpaired t - , ns = not significant , * P ≤ 0 . 05 .

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

J Axonal area occupied by mitochondria in Yme1l-/- neurons relative to WT controls. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM. Unpaired t-test, ns = not significant, *P ≤ 0.05.