F Luciferase Complementation Imaging ( LCI ) to detect interaction between OsCIPK7 - nluc OsCBL7 - cluc or OsCBL8 - cluc , with co - expression of N1 - 54 : mVenus : OsCRT3 ( mVenus - OsCRT3 ) or GFP , respectively . of luciferase activity was measured shown . Values are mean ± SD . At least 6 leaves were used for each sample . * P < 0 . 05 , Student ' s t - . Experiments were repeated at least twice with similar results .

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F Luciferase Complementation Imaging assay (LCI) to detect the interaction between OsCIPK7-nluc and OsCBL7-cluc or OsCBL8-cluc, with co-expression of N1-54:mVenus:OsCRT3 (mVenus-OsCRT3) or GFP, respectively. Quantification of luciferase activity was measured and shown. Values are mean ± SD. At least 6 leaves were used for each sample. *P < 0.05, Student's t-test. Experiments were repeated at least twice with similar results.


G Immunoblot confirming expression of OsCIPK7 , OsCBL7 , OsCBL8 , OsCRT3 in ( F ) respectively .

G Immunoblot confirming the expression of OsCIPK7, OsCBL7, OsCBL8, and OsCRT3 in (F) respectively.


H Pull - down showing that interaction between OsCIPK7 OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3 . MBP was used as a negative control . Relative expression was conducted using Image J software .

O O O O O O O O O O O O O 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 Pull-down assay showing that the interaction between OsCIPK7 and OsCBL7 or OsCBL8 was weakened with increasing amounts of OsCRT3. MBP was used as a negative control. Relative expression analysis was conducted using Image J software.


A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro . loaded is indicated by red arrows . Autoradiograph of γ - 32P - labeled OsCIPK7 indicates autophosphorylation of OsCIPK7 .

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A OsCIPK7 showed autophosphorylation activity but could not phosphorylate OsCRT3 in vitro. The loaded protein is indicated by red arrows. Autoradiograph of γ-32P-labeled OsCIPK7 indicates autophosphorylation of OsCIPK7.


B OsCIPK7 didn ' t phosphorylate OsCBL7 or OsCBL8 in vitro . Loaded stained with Coomassie brilliant blue ( CBB ) are shown in top panel indicated by red arrows . Autoradiograph of γ - 32P - labeled myelin basic OsCIPK7 are shown in bottom panel .

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B OsCIPK7 didn't phosphorylate OsCBL7 or OsCBL8 in vitro. Loaded proteins stained with Coomassie brilliant blue (CBB) are shown in the top panel and indicated by red arrows. Autoradiograph of γ-32P-labeled myelin basic protein and OsCIPK7 are shown in the bottom panel.


C kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 . SDS - PAGE gel showing CBB - stained OsCRT3 , myelin basic , OsCIPK7 ( top panel ) . γ - 32P - labeled myelin basic OsCIPK7 substrate phosphorylation ( bottom panel ) . amount of OsCRT3 added was 0 μg , 0 . 2 μg , 0 . 4 μg , 0 . 6 μg , 0 . 8 μg in SDS - PAGE gel lanes , respectively .

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C The kinase activity OsCIPK7 was enhanced with increasing concentrations of OsCRT3 protein. SDS-PAGE gel showing CBB-stained OsCRT3, myelin basic protein, and OsCIPK7 (top panel). γ-32P-labeled myelin basic protein and OsCIPK7 substrate phosphorylation (bottom panel). The amount of OsCRT3 added was 0 μg, 0.2 μg, 0.4 μg, 0.6 μg, 0.8 μg in the SDS-PAGE gel lanes, respectively.


( a ) Lysates extracted from SVZ of three different Ctrl FIP200GFAP cKO mice treated with chloroquine , then analyzed by western blot using antibodies to LC3 ( top ) , p62 ( middle ) or vinculin ( bottom ) .

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(a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).


( b ) Immunofluorescence for p62 DAPI in SVZ DG of Ctrl FIP200GFAP cKO mice at P28 ( n = 3 p62 for each ) . Dotted lines indicate boundaries of SVZ granular zone ( GZ ) . arrows arrowheads mark larger smaller p62 + , respectively , in striatum ( ST ) SVZ .

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(b) Immunofluorescence for p62 and DAPI in the SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 (n = 3 p62 for each). Dotted lines indicate boundaries of the SVZ and granular zone (GZ). The arrows and arrowheads mark larger and smaller p62+ aggregates, respectively, in the striatum (ST) and SVZ.


( c ) of mitochondria in SVZ tissue from Ctrl FIP200GFAP cKO mice at P28 P56 ( n = 2 mice for each , > 30 for each mouse ) . Arrows mark mitochondria .

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(c) TEM of mitochondria in SVZ tissue from Ctrl and FIP200GFAP cKO mice at P28 and P56 (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria.


( d ) of mitochondria in cultured neurospheres from Ctrl FIP200GFAP cKO mice ( n = 2 mice for each , > 30 for each mouse ) . Arrows mark mitochondria .

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(d) TEM of mitochondria in cultured neurospheres from Ctrl and FIP200GFAP cKO mice (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria.


( e , f ) DHE DAPI in dentate gyrus ( e ) SVZ ( f ) of Ctrl FIP200GFAP cKO mice at P28 ( n = 5 mice for each ) . Dotted lines indicate boundaries of GZ ( e ) SVZ ( f ) . Arrows mark in SGZ ( e ) SVZ ( f ) , arrowheads mark in surround regions ( GZ in e ST in f ) . LV , lateral ventricle ; ML , molecular layer . Full - blots are presented in Supplementary Figure 11 .

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(e,f) DHE and DAPI fluorescence in the dentate gyrus (e) and SVZ (f) of Ctrl and FIP200GFAP cKO mice at P28 (n = 5 mice for each). Dotted lines indicate the boundaries of the GZ (e) and SVZ (f). Arrows mark cells in the SGZ (e) and SVZ (f), and arrowheads mark cells in surround regions (the GZ in e and the ST in f). LV, lateral ventricle; ML, molecular layer. Full-length blots are presented in Supplementary Figure 11.


( a ) Hematoxylin eosin of dentate gyrus ( DG ) SVZ from Ctrl FIP200GFAP cKO mice at P28 . Lines indicate boundaries of DG SVZ . Arrows mark in SVZ .

(a) Hematoxylin and eosin staining of the dentate gyrus (DG) and SVZ from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark cells in the SVZ.


( b - e ) Immunofluorescence for GFAP , nestin or SOX2 DAPI in DG ( b , c ) from Ctrl FIP200GFAP cKO mice at P28 . boxed areas in c - e are shown in more detail in insets ( c ) . Dotted lines indicate boundaries of granular zone ( GZ ) SGZ ( b ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( b , c ) , arrowheads mark GFAP + nestin − GFAP + SOX2 − astrocytes .

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(b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the DG (b,c) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in insets (c). Dotted lines indicate boundaries of the granular zone (GZ) and SGZ (b). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.


( b - e ) Immunofluorescence for GFAP , nestin DAPI in SVZ ( d , e ) from Ctrl FIP200GFAP cKO mice at P28 . boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of SVZ ( d , e ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( b , c ) , arrowheads mark GFAP + nestin − GFAP + SOX2 − astrocytes .

(b-e) Immunofluorescence for GFAP, nestin and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.


( b - e ) Immunofluorescence for GFAP , nestin or SOX2 DAPI in SVZ ( d , e ) from Ctrl FIP200GFAP cKO mice at P28 . boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of SVZ ( d , e ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( b , c ) , arrowheads mark GFAP + nestin − GFAP + SOX2 − astrocytes .

(b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.


( f ) Immunofluorescence for PSA - NCAM in RMS ( boxed areas ) , SVZ DG of Ctrl FIP200GFAP cKO mice at P28 . Lines indicate boundaries of DG SVZ . Arrows mark PSA - NCAM + in RMS , SVZ DG .

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(f) Immunofluorescence for PSA-NCAM in the RMS (boxed areas), SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark PSA-NCAM+ cells in the RMS, SVZ and DG.


( g ) Immunofluorescence for NeuN in olfactory bulb ( OB ) DG of Ctrl FIP200GFAP cKO mice at P31 . Lines indicate layers of OB boundaries of DG . Boxed areas in top panels are shown in more detail for of NeuN in insets . CC , corpus callosum ; E , ependymal ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum ; IGL , internal granular layer ; MCL , mitral layer ; EPL , external plexiform layer ; GL , glomerular layer .

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(g) Immunofluorescence for NeuN in the olfactory bulb (OB) and DG of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the layers of the OB and boundaries of the DG. Boxed areas in top panels are shown in more detail for staining of NeuN in insets. CC, corpus callosum; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer.


( a - e ) ( 1st ) secondary ( 2nd ) neurospheres from Ctrl FIP200GFAP cKO mice at P0 P28 . Representative P0 phase contrast are shown in a . Mean ± s . e . m . of number ( b , d ) ( c , e ) of secondary neurospheres from three independent experiments are shown ( n = 3 mice for each ) .

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(a-e) Primary (1st) and secondary (2nd) neurospheres from Ctrl and FIP200GFAP cKO mice at P0 and P28. Representative P0 phase contrast images are shown in a. Mean ± s.e.m. of number (b,d) and size (c,e) of primary and secondary neurospheres from three independent experiments are shown (n = 3 mice for each).


( f ) Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) DAPI of SVZ dentate gyrus ( DG ) from Ctrl FIP200GFAP cKO mice at P28 . Lines indicate boundaries of SVZ . Numbered boxed areas are shown in more detail as insets for of TUNEL DAPI in SVZ DG .

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(f) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DAPI staining of the SVZ and dentate gyrus (DG) from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the SVZ. Numbered boxed areas are shown in more detail as insets for staining of TUNEL and DAPI in the SVZ and DG.


( g ) Short - term BrdU incorporation in SVZ DG of Ctrl FIP200GFAPcKO mice at P28 . Dotted lines indicate boundaries of SVZ granular zone ( GZ ) . Boxed areas are shown in more detail in insets . Lines indicate boundaries of SVZ GZ . Arrows mark BrdU + . CC , corpus callosum ; LV , lateral ventricle ; ST , striatum . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 .

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(g) Short-term BrdU incorporation in the SVZ and DG of Ctrl and FIP200GFAPcKO mice at P28. Dotted lines indicate the boundaries of the SVZ and granular zone (GZ). Boxed areas are shown in more detail in the insets. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001.


( h ) Long - term retention of BrdU in SVZ dentate gyrus of Ctrl FIP200GFAP cKO mice at P31 . Lines indicate boundaries of SVZ GZ . Arrows mark BrdU + . CC , corpus callosum ; LV , lateral ventricle ; ST , striatum . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 .

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(h) Long-term retention of BrdU in the SVZ and dentate gyrus of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001.


( a ) Lysates extracted from neurospheres of Ctrl , FIP200GFAP cKO , Trp53GFAP cKO 2cKO mice analyzed by western blot using antibodies to p53 actin .

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(a) Lysates extracted from neurospheres of Ctrl, FIP200GFAP cKO, Trp53GFAP cKO and 2cKO mice and analyzed by western blot using antibodies to p53 and actin.


( b ) Mean ± s . e . m . of relative mRNA level ( normalized to Ctrl mice as 1 ) of Cdkn1a , Bbc3 , Fas , Pax6 Stat3 in neurospheres of Ctrl , FIP200GFAP cKO , 2cKO Trp53GFAP cKO mice are shown ( n = 3 mice for each ) .

(b) Mean ± s.e.m. of relative mRNA level (normalized to Ctrl mice as 1) of Cdkn1a, Bbc3, Fas, Pax6 and Stat3 in neurospheres of Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each).


( c , d ) Mean ± s . e . m . of number ( c ) ( d ) of secondary neurospheres from Ctrl , FIP200GFAP cKO , 2cKO Trp53GFAP cKO mice at P28 ( n = 3 mice for each ) .

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(c,d) Mean ± s.e.m. of the number (c) and size (d) of primary and secondary neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice at P28 (n = 3 mice for each).


( e - h ) of immunofluorescence or on frozen - sectioned neurospheres for nestin , Ki67 , TUNEL , PAX6 , phosphorylated STAT3 ( pSTAT3 ) DAPI , as indicated . Mean ± s . e . m . of of Ki67 + ( e ) , BrdU + ( f ) TUNEL + ( g ) in neurospheres from Ctrl , FIP200GFAP cKO , 2cKO Trp53GFAP cKO mice are shown ( n = 3 mice for each , counting of > 500 for each mouse ) . Representative are shown in h . * P 0 . 01 . Full - blots are presented in Supplementary Figure 11 .

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(e-h) Analysis of immunofluorescence or staining on frozen-sectioned neurospheres for nestin, Ki67, TUNEL, PAX6, phosphorylated STAT3 (pSTAT3) and DAPI, as indicated. Mean ± s.e.m. of the percentage of Ki67+ (e), BrdU+ (f) and TUNEL+ (g) cells in neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each, counting of >500 cells for each mouse). Representative images are shown in h. *P 0.01. Full-length blots are presented in Supplementary Figure 11.


( a - c ) Hematoxylin eosin of dentate gyrus dentate gyrus ( DG ) of P28mice ( a ) . Arrows mark in SVZ . Mean ± s . e . m . of SVZ cellularity ( b ) DG ( c ) per section are shown . Dotted lines indicate boundaries of SVZ DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

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(a-c) Hematoxylin and eosin staining of the dentate gyrus and dentate gyrus (DG) of P28mice (a). Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( d - m ) Immunofluorescence of DG ( d - h ) of P28mice ( d , e , i , j ) . Boxed areas are shown in more detail in insets ( e , j ) / or panels below ( i , j ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( d , e ) , arrowheads mark GFAP + nestin − GFAP + SOX2 − astrocytes . Mean ± s . e . m . of number of GFAP + nestin + GFAP + SOX2 + radial glia ( f , h ) , NSCs ( k , m ) , GFAP + nestin − astrocytes ( g , l ) per section are shown . Dotted lines indicate boundaries of SVZ DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

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(d-m) Immunofluorescence of the DG (d-h) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( d - m ) Immunofluorescence of SVZ ( i - m ) of P28mice ( d , e , i , j ) . Boxed areas are shown in more detail in insets ( e , j ) / or panels below ( i , j ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( d , e ) , arrowheads mark GFAP + nestin − GFAP + SOX2 − astrocytes . Mean ± s . e . m . of number of GFAP + nestin + GFAP + SOX2 + radial glia ( f , h ) , NSCs ( k , m ) , GFAP + nestin − astrocytes ( g , l ) per section are shown . Dotted lines indicate boundaries of SVZ DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

(d-m) Immunofluorescence of the SVZ (i-m) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( n , o ) Mean ± s . e . m . of number of TUNEL + per 100 SVZ ( n ) or per 1 mm2 DG ( o ) of P28 mice .

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(n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28 mice.


( p - r ) BrdU retention in SVZ DG of P31mice . BrdU + are marked by arrows ( p ) . Mean ± s . e . m . of number of BrdU + per section are shown ( q , r ) . Dotted lines indicate boundaries of SVZ DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 .

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(p-r) BrdU retention in the SVZ and DG of P31mice. BrdU+ cells are marked by arrows (p). Mean ± s.e.m. of the number of BrdU+ cells per section are shown (q,r). Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001


( s , t ) Mean ± s . e . m . of of GFAP + nestin + BrdU + of total BrdU retained in SVZ ( s ) SGZ ( t ) of P31mouse . n = 5 mice , ≥ 4 sections per mouse , > 500 counted per mouse in n , > 20 BrdU + counted per mouse .

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(s,t) Mean ± s.e.m. of percentage of GFAP+nestin+BrdU+ cells of total BrdU retained cells in the SVZ (s) and SGZ (t) of P31mouse. n = 5 mice, ≥4 sections per mouse, >500 cells counted per mouse in n, >20 BrdU+ cells counted per mouse.


( a - f ) Immunofluorescence for PSA - NCAM ( a - c ) or NeuN ( d - f ) in dentate gyrus ( DG ) , SVZ , RMS olfactory bulb ( OB ) of P28mice . Lines indicate boundaries of DG ( a , d ) , SVZ ( a ) , layers of OB ( d ) . Boxed areas in a indicate RMS . Arrows mark PSA - NCAM + . Mean ± s . e . m . of number of PSA - NCAM + ( b , c ) or NeuN + ( e , f ) per section are shown . LV , lateral ventricle ; ML , molecular layer ; IGL , internal granular layer ; MCL , mitral layer ; EPL , external plexiform layer ; GL , glomerular layer . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28mice. Lines indicate boundaries of the DG (a,d), SVZ (a), and layers of the OB (d). Boxed areas in a indicate the RMS. Arrows mark PSA-NCAM+ cells. Mean ± s.e.m. of the number of PSA-NCAM+ cells (b,c) or NeuN+ cells (e,f) per section are shown. LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.


( g - i ) Immunofluorescence of BrdU retention in DG OB of P31 mice . Boxed areas in left column of g are shown in more detail in right column . Dotted lines indicate boundaries of DG ( left ) GZ ( right ) . Arrows mark NeuN + BrdU + arrowheads mark NeuN − BrdU + in DG . Mean ± s . e . m . of of NeuN + BrdU + of total BrdU retained in DG ( h ) OB ( i ) are shown . ( j , k ) Mean ± s . e . m . of of GFAP + nestin − BrdU + among total BrdU + retained in SVZ ( j ) SGZ ( k ) .

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

(g-i) Immunofluorescence of BrdU retention cells in the DG and OB of P31 mice. Boxed areas in left column of g are shown in more detail in right column. Dotted lines indicate the boundaries of the DG (left) and GZ (right). Arrows mark NeuN+BrdU+ cells and arrowheads mark NeuN−BrdU+ cells in the DG. Mean ± s.e.m. of the percentage of NeuN+BrdU+ cells of total BrdU retained cells in the DG (h) and OB (i) are shown. (j,k) Mean ± s.e.m. of percentage of GFAP+nestin−BrdU+ cells among total BrdU+ retained cells in the SVZ (j) and SGZ (k).


( l , m ) DHE DAPI at P28 in DG ( l ) SVZ ( m ) from FIP200GFAP cKO mice with or without NAC treatment , 2cKO mice Trp53GFAP cKO mice . Dotted lines indicate boundaries of granular zone ( GZ ) ( l ) SVZ ( m ) . Arrows mark in SGZ ( l ) SVZ ( m ) arrowheads mark in surrounding regions ( GZ in l striatum ( ST ) in m ) . n = 5 mice , ≥ 4 sections per mouse , > 20 ( h , j , k ) or > 200 ( i ) BrdU + counted per mouse . cKO ( n ) , NAC non - responder cKO mice ; LV , lateral ventricle ; ML , molecular layer ; IGL , internal granular layer ; MCL , mitral layer ; EPL , external plexiform layer ; GL , glomerular layer . 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(l,m) DHE and DAPI fluorescence at P28 in the DG (l) and SVZ (m) from FIP200GFAP cKO mice with or without NAC treatment, 2cKO mice and Trp53GFAP cKO mice. Dotted lines indicate the boundaries of the granular zone (GZ) (l) and SVZ (m). Arrows mark cells in the SGZ (l) and SVZ (m) and arrowheads mark cells in surrounding regions (the GZ in l and the striatum (ST) in m). n = 5 mice, ≥4 sections per mouse, >20 (h,j,k) or >200 (i) BrdU+ cells counted per mouse. cKO(n), NAC non-responder cKO mice; LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.


( a - c ) Hematoxylin eosin ( a ) of SVZ dentate gyrus ( DG ) of P28mice treated by NAC or vehicle ( Veh ) control . Dotted lines indicate boundaries for SVZ DG . Arrows mark in SVZ .

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

(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ.


( a - c ) Hematoxylin eosin ( a ) of SVZ dentate gyrus ( DG ) of P28 mice treated by NAC or vehicle ( Veh ) control . Dotted lines indicate boundaries for SVZ DG . Arrows mark in SVZ . Mean ± s . e . m . of SVZ cellularity ( b ) DG ( c ) per section are shown .

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

(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28 mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown.


( d - m ) Immunofluorescence of DG ( d - h ) SVZ ( i - m ) of P28 mice treated by NAC or vehicle control . Representative are shown in d , g ( DG ) k , m ( SVZ ) . Dotted lines indicate boundaries of granular zone ( GZ ) ( d , g ) SVZ ( k , m ) . Boxed areas in g , k , m are shown in more detail in insets ( g , m ) / or panels below ( k , m ) . Lines indicate boundaries of GZ ( d , g ) SVZ ( k , m ) . Arrows mark GFAP + nestin + GFAP + SOX2 + NSCs with radial glial ( d , g ) , arrowheads mark GFAP + nestin − GFAP + SOX2 − astrocytes . Mean ± s . e . m . of number of GFAP + nestin + GFAP + SOX2 + radial glia ( e , h ) , NSCs ( i , l ) , GFAP + nestin − astrocytes ( f , j ) per section are shown . ( n , o ) Mean ± s . e . m . of number of TUNEL + per 100 SVZ ( n ) or per 1 mm2 DG ( o ) of P28mice treated by NAC or vehicle control . n = 5 mice , ≥ 4 sections per mouse , > 500 counted per NAC in n . cKO ( n ) , NAC non - responder cKO mice ; E , ependymal ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . * * P 0 . 01 .

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

(d-m) Immunofluorescence of the DG (d-h) and SVZ (i-m) of P28 mice treated by NAC or vehicle control. Representative images are shown in d,g (DG) and k,m (SVZ). Dotted lines indicate boundaries of the granular zone (GZ) (d,g) and SVZ (k,m). Boxed areas in g,k,m are shown in more detail in insets (g,m) and/or panels below (k,m). Lines indicate boundaries of the GZ (d,g) and SVZ (k,m). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,g), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (e,h), and NSCs (i,l), and GFAP+nestin− astrocytes (f,j) per section are shown. (n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28mice treated by NAC or vehicle control. n = 5 mice, ≥4 sections per mouse, >500 cells counted per NAC in n. cKO(n), NAC non-responder cKO mice; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. **P 0.01.


( a - f ) Immunofluorescence for PSA - NCAM ( a - c ) or NeuN ( d - f ) in dentate gyrus ( DG ) , SVZ , RMS olfactory bulb ( OB ) of P28 mice treated with NAC or vehicle ( Veh ) control . Representative are shown for PSA - NCAM ( a ) NeuN ( d ) . Dotted lines indicate boundaries of DG ( a , d ) , SVZ ( a ) layers of OB ( d ) . Arrows mark PSA - NCAM + in a . Mean ± s . e . m . of number of PSA - NCAM + in SVZ ( b ) DG ( normalized to 1 mm2 ) ( c ) , number of NeuN + in DG ( e ) IGL of OB ( per mm2 ) ( f ) per section are shown ( n = 5 mice for each ; ≥ 4 sections for each mouse ) . cKO ( n ) , NAC non - responder cKO mice ; GZ , granular zone ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum ; IGL , internal granular layer ; MCL , mitral layer ; EPL , external plexiform layer ; GL , glomerular layer . * * P 0 . 01 .

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

(a-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28 mice treated with NAC or vehicle (Veh) control. Representative images are shown for PSA-NCAM (a) and NeuN (d). Dotted lines indicate boundaries of the DG (a,d), SVZ (a) and layers of the OB (d). Arrows mark PSA-NCAM+ cells in a. Mean ± s.e.m. of the number of PSA-NCAM+ cells in SVZ (b) and DG (normalized to 1 mm2 area) (c), and the number of NeuN+ cells in DG (e) and IGL of OB (per mm2) (f) per section are shown (n = 5 mice for each; ≥4 sections for each mouse). cKO(n), NAC non-responder cKO mice; GZ, granular zone; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. **P 0.01.


A Lysates of HeLa stably expressing GFP alone or GFP - FXR1 were subjected to immunoprecipitation using GFP - Trap beads ( GFP - IP ) , analysed by Western blot quantified ( shown a mean value , * P < 0 . 05 , * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (shown a mean value, *P < 0.05, **P < 0.01; N = 3).


B Lysates of HeLa stably expressing GFP alone or 3xGFP - Nup85 were immunoprecipitated using GFP - Trap beads ( GFP - IP ) , analysed by Western blot quantified ( SE , short exposure , LE , long exposure ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of HeLa cells stably expressing GFP alone or 3xGFP-Nup85 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (SE, short exposure, LE, long exposure) (shown a mean value, *P < 0.05; N = 3).


C Immunoprecipitation from HEK293T lysates using FXR1 antibody or IgG analysed by Western blot . arrow points to heavy chain of IgG ( IgG HC ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O 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 Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot. The arrow points to the heavy chain of IgG (IgG HC) (shown a mean value, *P < 0.05; N = 3).


D HeLa were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy for lamin B receptor ( LBR ) to label NE , FXR1 .

O O O O O O O O O O O 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 HeLa cells were treated with indicated siRNAs, synchronized by double thymidine block, and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor (LBR) to label the NE, and FXR1.


E HeLa stably expressing GFP - FXR1 were analysed by immunofluorescence microscopy for GFP mAb414 , which labels FG - Nups . magnified framed regions are shown in corresponding numbered panels . arrowheads indicate NE localization of GFP - FXR1 .

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

E HeLa cells stably expressing GFP-FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414, which labels FG-Nups. The magnified framed regions are shown in the corresponding numbered panels. The arrowheads indicate NE and cytoplasmic localization of GFP-FXR1.


F HeLa stably expressing GFP - Nup107 were synchronized by double thymidine block released for 12 hours , permeabilized with Triton / SDS or digitonin for antibodies to access or cytoplamsic side of nucleus , respectively , analysed by immunofluorescence microscopy .

O O O O O O O O O O O O O O O O O O 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 HeLa cells stably expressing GFP-Nup107 were synchronized by double thymidine block and released for 12 hours, permeabilized with Triton/SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus, respectively, and analysed by immunofluorescence microscopy.


A HeLa were treated with indicated siRNAs , synchronized by double thymidine block release for 12 hours analysed by immunofluorescence microscopy . magnified framed regions are shown in corresponding numbered panels .

O O O O O O O O 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 HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.


B HeLa stably expressing GFP , GFP - FXR1 wild type ( WT ) GFP - FXR1 mutated in recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with indicated siRNAs , synchronized by double thymidine block , released for 24 hours then analysed by immunofluorescence microscopy . of with nucleoporin granules was quantified , 1000 were analysed for each graph ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 , N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, 1000 cells were analysed for each graph (mean ±SD, **P < 0.01; ***P < 0.001, N = 3).


HeLa were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . Nups present in different NPC subcomplexes are depicted in color code corresponding to NPC scheme shown on right . Additional or complementary representative channels of depicted in ( C )

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

HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C)


HeLa were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . of FG - Nups labelled by mAb414 ( D ) , RanBP2 ( E ) GFP - Nup107 ( F ) was quantified . 1800 were analysed for each graph ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) .

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

HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nuclear intensity of FG-Nups labelled by mAb414 (D), RanBP2 (E) and GFP-Nup107 (F) was quantified. 1800 cells were analysed for each graph (mean ±SD, *P < 0.05; **P < 0.01; N = 3).


G , H Asynchronously proliferating U2OS were treated with indicated siRNAs analysed by immunofluorescence microscopy . of with nucleoporin granules ( G ) was quantified of FG - Nups labelled by mAb414 ( H ) was quantified . 1600 were analysed in ( G ) 2100 were analysed in ( H ) ( mean ± SD , * P < 0 . 05 ; * * * P < 0 . 001 ; N = 3 ) .

O O 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

G, H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules (G) was quantified and nuclear intensity of FG-Nups labelled by mAb414 (H) was quantified. 1600 cells were analysed in (G) and 2100 cells were analysed in (H) (mean ±SD, *P < 0.05; *** P<0.001; N = 3).


A HeLa were treated with indicated siRNAs , synchronized by double thymidine block , released for 24 hours analysed by immunofluorescence microscopy . magnified framed regions are shown in corresponding numbered panels . Arrowheads point to blebs observed in FXR1 - deficient .

O O O O O O O O O O O O O O O O O O O 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 HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. Arrowheads point to nuclear blebs observed in FXR1-deficient cells.


B , HeLa stably expressing GFP , GFP - FXR1 wild type ( WT ) GFP - FXR1 mutated in recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with indicated siRNAs , synchronized by double thymidine block , released for 24 hours analysed by Western blot ( B )

B, HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by Western blot (B)


C HeLa stably expressing GFP , GFP - FXR1 wild type ( WT ) GFP - FXR1 mutated in recognized by FXR1 siRNA - 1 ( GFP - FXR1 - MUT - siRNA1 ) were treated with indicated siRNAs , synchronized by double thymidine block , released for 24 hours analysed by immunofluorescence microscopy ( C ) . of with irregular nuclei was quantified , 1000 were analysed ( mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy (C). The percentage of cells with irregular nuclei was quantified, 1000 cells were analysed (mean ±SD, **P < 0.01, ***P < 0.001; N = 3).


HeLa stably expressing marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . Time from prophase till anaphase ( D ) , from prophase till metaphase ( E ) , from metaphase till anaphase ( F ) from anaphase till decondensation ( G ) was quantified .

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

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Time from prophase till anaphase (D), from prophase till metaphase (E), from metaphase till anaphase (F) and from anaphase till chromatin decondensation (G) was quantified.


HeLa stably expressing marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . selected frames of movies are depicted time is shown in minutes ( H ) . Arrowheads point to blebs appearing during expansion of FXR1 - deficient .

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

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The selected frames of the movies are depicted and time is shown in minutes (H). Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1-deficient cells.


HeLa stably expressing marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . of daughter with irregular nuclei was quantified in ( I )

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

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Percentage of daughter cells with irregular nuclei was quantified in (I)


HeLa stably expressing marker histone H2B labelled with mCherry were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . time from anaphase till blebs was quantified in ( J ) .

HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. time from anaphase till nuclear blebs was quantified in (J).


A - D HeLa stably expressing GFP - Nup107 were treated with indicated siRNAs , synchronized by double thymidine block , released analysed by live video spinning disk confocal microscopy ( A ) . selected frames of movies are depicted time is shown in minutes . onset of anaphase is indicated . magnified framed regions with time indicated in minutes are shown in ( B ) . arrowheads point to GFP - NUP107 granules appearing during expansion of control FXR1 - deficient , yellow arrowheads point to fussion events of GFP - NUP107 granules with NE in control . of with GFP - Nup107 granules was quantified in ( C ) . Time from anaphase till GFP - Nup107 granule formation was quantified in ( D ) . 57 were analysed ( mean ± SD , * P < 0 . 05 ; ns = non - significant ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D HeLa cells stably expressing GFP-Nup107 were treated with indicated siRNAs, synchronized by double thymidine block, released and analysed by live video spinning disk confocal microscopy (A). The selected frames of the movies are depicted and time is shown in minutes. The onset of anaphase is indicated. The magnified framed regions with time indicated in minutes are shown in (B). White arrowheads point to the cytoplasmic GFP-NUP107 granules appearing during nuclear expansion of control and FXR1-deficient cells, yellow arrowheads point to the fussion events of GFP-NUP107 granules with NE in control cells. The percentage of cells with cytoplasmic GFP-Nup107 granules was quantified in (C). Time from anaphase till GFP-Nup107 cytoplasmic granule formation was quantified in (D). 57 cells were analysed (mean ±SD, *P < 0.05; ns = non-significant; N = 3).


A HeLa stably expressing GFP - Nup133 were synchronized by double thymidine block , released for 12 hours , treated or not with NaAsO2 to induce stress granule formation with 1 , 6 Hexanediol , analysed by immunofluorescence microscopy . magnified framed regions are shown in corresponding numbered panels .

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

A HeLa cells stably expressing GFP-Nup133 were synchronized by double thymidine block, released for 12 hours, treated or not with NaAsO2 to induce stress granule formation and with 1,6 Hexanediol, and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.


B , C HeLa stably expressing GFP - Nup133 were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with or without 1 , 6 Hexanediol analysed by immunofluorescence microscopy . magnified framed regions are shown in corresponding numbered panels . of with GFP - Nup133 granules was quantified in ( C ) , 3100 were analysed ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; N = 3 ) .

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

B, C HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with or without 1,6 Hexanediol and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (C), 3100 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; ****P < 0.0001; N = 3).


A HeLa stably expressing GFP alone or GFP - FXR1 were immunoprecipitated using GFP - Trap beads ( GFP - IP ) , analysed by Western blot quantified ( mean , * P < 0 . 05 ; N = 3 ) .

A HeLa cells stably expressing GFP alone or GFP-FXR1 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; N = 3).


B , C HeLa were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . Arrowheads indicate blebbed regions of nuclei . of interphasic with Nup granules was quantified ( C ) , 900 were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) .

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B, C HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Arrowheads indicate blebbed regions of nuclei. The percentage of interphasic cells with Nup granules was quantified (C), 900 cells were analysed (mean ±SD, ***P < 0.001; N = 3).


D Lysates of HeLa stably expressing GFP alone or GFP - FXR1 were subjected to immunoprecipitation using GFP - Trap beads ( GFP - IP ) , analysed by Western blot quantified ( mean , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; **P < 0.01; N = 3).


E , F HeLa were treated with indicated siRNAs , synchronized by double thymidine block released for 12 hours analysed by immunofluorescence microscopy for FXR1 mAb414 . magnified framed regions are shown in corresponding numbered panels in ( E ) . of with Nup granules was quantified in ( F ) , 2700 were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) .

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

E, F HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414. The magnified framed regions are shown in the corresponding numbered panels in (E). The percentage of cells with cytoplasmic Nup granules was quantified in (F), 2700 cells were analysed (mean ±SD, ***P < 0.001; N = 3).


A , B HeLa stably expressing GFP - Nup133 were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with nocodazole to induce granule formation washed - out as indicated analysed by immunofluorescence microscopy . of with GFP - Nup133 granules was quantified in ( B ) , 5200 were analysed ( mean ± SD , * * P < 0 . 01 ; * * * P < 0 . 001 ; N = 3 ) .

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

A, B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as indicated and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (B), 5200 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; N = 3).


C - E HeLa stably expressing GFP - Nup107 were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours , treated with nocodazole to induce granule formation washed - out as in ( A ) analysed by live video spinning disk confocal microscopy . selected frames of movies are depicted time is shown in minutes . magnified framed regions are depicted in lower rows . arrowheads point to GFP - Nup107 - positive granules . Yellow arrowheads point to granules undergoing fusion events . of with fusion / fission events of GFP - Nup107 granules was quantified in ( D ) , 815 were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) . number of fusion / fission events per was quantified in ( E ) , 815 were analysed ( mean ± SD ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E HeLa cells stably expressing GFP-Nup107 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as in (A) and analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted and time is shown in minutes. The magnified framed regions are depicted in the lower rows. White arrowheads point to individual GFP-Nup107-positive granules. Yellow arrowheads point to the granules undergoing fusion events. The percentage of cells with fusion/fission events of GFP-Nup107 granules was quantified in (D), 815 cells were analysed (mean ±SD, **P < 0.01; N = 3). The number of fusion/fission events per cell was quantified in (E), 815 cells were analysed (mean ±SD; N = 3).


Human FXS - derived fibroblasts ( FXS - 1 , FXS - 2 ) control human fibroblasts were synchronized in early G1 by Monastrol release analysed by Western blot ( A )

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

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by Western blot (A)


Human FXS - derived fibroblasts ( FXS - 1 , FXS - 2 ) control human fibroblasts were synchronized in early G1 by Monastrol release analysed by immunofluorescence microscopy Examples of Nup localization defects are shown in ( B )

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

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy Examples of Nup localization defects are shown in (B)


Human FXS - derived fibroblasts ( FXS - 1 , FXS - 2 ) control human fibroblasts were synchronized in early G1 by Monastrol release analysed by immunofluorescence microscopy of with Nup granules was quantified in ( C ) , 283 were analysed ( mean ± SD , * * * P < 0 . 001 ; N = 3 ) .

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

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup granules was quantified in (C), 283 cells were analysed (mean ±SD, ***P < 0.001; N = 3).


Human FXS - derived fibroblasts ( FXS - 1 , FXS - 2 ) control human fibroblasts were synchronized in early G1 by Monastrol release analysed by immunofluorescence microscopy examples of architecture defects are shown in ( D ) .

Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy examples of nuclear architecture defects are shown in (D).


Human induced pluripotent ( iPSCs ) derived from a FXS ( FXS - iPSC ) isogenic rescue ( C1 _ 2 - iPSC ) were analysed by immunofluorescence

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

Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy


Human induced pluripotent ( iPSCs ) derived from a FXS ( FXS - iPSC ) isogenic rescue ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopy of with Nup133 granules was quantified in ( F ) , 5500 were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) .

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

Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy The percentage of cells with cytoplasmic Nup133 granules was quantified in (F), 5500 cells were analysed (mean ±SD, **P < 0.01; N = 3).


Human induced pluripotent ( iPSCs ) derived from a FXS ( FXS - iPSC ) isogenic rescue ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopy Examples of co - localization events of re - expressed FMRP Nup133 are shown in ( G ) .

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

Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy Examples of co-localization events of re-expressed FMRP and Nup133 are shown in (G).


H , I Mouse Embryonic Fibroblasts ( MEFs ) derived from Fmr1 knock - out ( KO ) mice wild type controls were synchronized in early G1 by Monastrol release analysed by immunofluorescence microscopy ( H ) . of with nucleoporin granules was quantified in ( I ) , 2400 were analysed ( mean ± SD , * P < 0 . 05 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Mouse Embryonic Fibroblasts (MEFs) derived from the Fmr1 knock-out (KO) mice and wild type controls were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy (H). The percentage of cells with cytoplasmic nucleoporin granules was quantified in (I), 2400 cells were analysed (mean ±SD, *P < 0.05; N = 3).


A HeLa were transfected with import / export reporter plasmid XRGG - GFP , treated with indicated siRNAs synchronized in early G1 phase by Monastrol release . Dexamethasone was added for 3 hours to induce XRGG - GFP import . Following wash - out export of XRGG - GFP was analysed by live video spinning disk confocal microscopy . selected frames of movies are depicted in Figure EV5C . of XRGG - GFP over time was quantified in Figure EV5D quantifications of from 20 30 minutes time points are depicted in ( A ) , 199 were analysed ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 ) .

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A HeLa cells were transfected with the import/export reporter plasmid XRGG-GFP, treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release. Dexamethasone was added for 3 hours to induce XRGG-GFP nuclear import. Following wash-out the nuclear export of XRGG-GFP was analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted in Figure EV5C. The percentage of cytoplasmic XRGG-GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in (A), 199 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3).


B HeLa stably expressing GFP - Nup133 were treated with indicated siRNAs , synchronized by double thymidine block , released for 12 hours analysed by immunofluorescence microscopy . magnified framed regions are shown in corresponding numbered panels .

O O O O O O O O O O O O O O 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 HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.


C , D Asynchronously proliferating HeLa were treated with indicated siRNAs analysed by immunofluorescence microscopy ( C ) . of p - Rb positive was quantified in ( D ) , 2800 were analysed ( mean ± SD , * * P < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy (C). The percentage of p-Rb positive cells was quantified in (D), 2800 cells were analysed (mean ±SD, **P < 0.01; N = 3).


Asynchronously proliferating HeLa were treated with indicated siRNAs , incubated with EdU during 30min analysed by immunofluorescence microscopy . of p - Rb / or EdU positive was quantified in ( E ) , 2100 were analysed ( mean ± SD , * P < 0 . 05 ; * * P < 0 . 01 ; N = 3 )

Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. The percentage of p-Rb and/or EdU positive cells was quantified in (E), 2100 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3)


F Asynchronously proliferating HeLa were treated with indicated siRNAs , incubated with EdU during 30min analysed by immunofluorescence microscopy . of p - Rb / or cyclin B positive was quantified in (

O O O O O O O O O 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 Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. the percentage of p-Rb and/or cyclin B positive cells was quantified in (


A B16F10 melanoma with either mTRF2 overexpression or knockdown were inoculated into backs of immunocompetent mice . percentages of intratumoral CD11bHi GR1Hi MDSCs ( n = 10 mice ) , CD25 + Foxp3 + regulatory T ( Tregs ) , or CD25 + Foxp3 − activated T ( n = 5 mice per group ) among CD45 + were analyzed by - activated sorting ( FACS ) represented as box plots Data information : data are presented by Box plots Min to Max showing points with median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 ; Mann - Whitney .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B16F10 melanoma cells with either mTRF2 overexpression or knockdown were inoculated into the backs of immunocompetent mice. The percentages of intratumoral CD11bHi GR1Hi MDSCs (n = 10 mice), CD25+ Foxp3+ regulatory T cells (Tregs), or CD25+ Foxp3− activated T cells (n = 5 mice per group) among CD45+ cells were analyzed by fluorescence-activated cell sorting (FACS) and represented as box plots Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test.


TRF2 - overexpressing or - knockdown BJcl2 were inoculated into backs of immunodeficient mice in presence of Matrigel . Five days later , of immune infiltrating Matrigel plugs of CD11bHi GR1Hi MDSCs ( C ) were determined by FACS ( n = 5 mice per group ) .

TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later, the percentage of immune cells infiltrating the Matrigel plugs of CD11bHi GR1Hi MDSCs (C) were determined by FACS (n = 5 mice per group).


TRF2 - overexpressing or - knockdown BJcl2 were inoculated into backs of immunodeficient mice in presence of Matrigel . Five days later , intranuclear of pSTAT3 ( D ) were determined by FACS ( n = 5 mice per group ) .

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

TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later , the intranuclear staining of pSTAT3 (D) were determined by FACS (n = 5 mice per group).


TRF2 - overexpressing or - knockdown BJcl2 were inoculated into backs of immunodeficient mice in presence of Matrigel . Five days later , CD3 − NKp46 + IFN - γ + among CD3 − NKp46 + NK ( E ) were determined by FACS ( n = 5 mice per group ) .

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

TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later, CD3− NKp46+ and IFN-γ+ among CD3− NKp46+ NK cells (E) were determined by FACS (n = 5 mice per group).


Linear correlation plot of of IFN - γ + NK , mean of ( MFI ) of pSTAT3 for MDSCs , expression of TRF2 ( r2 = 0 , 5177 ; Pearson ' s correlation , p = 0 . 003 ) ( F ) Data information : data are presented by Box plots Min to Max showing points with median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 ; Mann - Whitney . Pearson correlation p values are indicated .

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

Linear correlation plot of the percentage of IFN- γ + NK cells, the mean of fluorescence intensity (MFI) of pSTAT3 for MDSCs, and the expression of TRF2 (r2= 0,5177; Pearson's correlation, p = 0.003) (F) Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. Pearson correlation and p values are indicated.


mRNA expression level in matrigel plugs of arg1 , pSTAT3 MFI TRF2 expression ( r2 = 0 , 7077 ; Pearson ' s correlation , p < 0 . 0001 ) ( G ) ; mRNA expression level in matrigel plugs of tgfβ , pSTAT3 MFI TRF2 expression ( r2 = 0 , 3669 ; Pearson ' s correlation , p = 0 . 0046 ) ( H ) mRNA expression level in matrigel plugs of il - 10 , pSTAT3 MFI TRF2 expression ( r2 = 0 , 7418 ; Pearson ' s correlation , p < 0 . 0001 ) ( I ) . Data information : data are presented by Box plots Min to Max showing points with median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 ; Mann - Whitney . Pearson correlation p values are indicated .

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

the mRNA expression level in the matrigel plugs of arg1, pSTAT3 MFI and TRF2 expression (r2= 0,7077; Pearson's correlation, p < 0.0001) (G); the mRNA expression level in the matrigel plugs of tgfβ, pSTAT3 MFI and TRF2 expression (r2= 0,3669; Pearson's correlation, p = 0.0046) (H) and the mRNA expression level in the matrigel plugs of il-10, pSTAT3 MFI and TRF2 expression (r2= 0,7418; Pearson's correlation, p < 0.0001) (I). Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. Pearson correlation and p values are indicated.


B16F10 melanoma with or without mTRF2 overexpression were inoculated into backs of immunocompetent mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) . volume was determined every 2 days using hemi - ellipsoid formula ( B ) . volume are presented in mm3 along time in days . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into the backs of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP). The tumor volume was determined every 2 days using hemi-ellipsoid formula (B). Tumor volume are presented in mm3 along the time in days. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


B16F10 melanoma with or without mTRF2 overexpression were inoculated into backs of immunocompetent mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) . On day 19 of growth experiment , percentages of GFP + infiltrating lungs were determined by FACS presented as number of GFP + / mg lung tissue ( C ) . Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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

B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into the backs of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP). On day 19 of the tumor growth experiment, the percentages of GFP+ cells infiltrating the lungs were determined by FACS and presented as the number of GFP+ cells/mg lung tissue (C). Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


On day 21 , lung metastasis was analyzed by counting macroscopic metastatic nodules ( E scale bars = 500µM .

On day 21, lung metastasis was analyzed by counting macroscopic metastatic nodules (E scale bars = 500µM.


B16F10 melanoma with or without mTRF2 overexpression were inoculated into tail vein of immunocompetent mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) every 3 days . On day 21 , lung metastasis was analyzed by hematoxylin eosin ( H & E ) F ) , scale bars = 500µM . Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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

B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into tail vein of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) every 3 days. On day 21, lung metastasis was analyzed by hematoxylin and eosin (H&E) staining F), scale bars = 500µM. Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


- infiltrating immune from tumors were analyzed by FACS : MDSCs CD25 + Foxp3 + Tregs ( G ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

Tumor-infiltrating immune cells from tumors were analyzed by FACS: MDSCs and CD25+ Foxp3+ Tregs (G) Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


- infiltrating immune from tumors were analyzed by FACS NK ( H ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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

Tumor-infiltrating immune cells from tumors were analyzed by FACS NK cells (H) Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


- infiltrating immune from tumors were analyzed by FACS : CD8 + T ( I ) . Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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Tumor-infiltrating immune cells from tumors were analyzed by FACS: CD8+ T cells (I). Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


BJcl2 with or without hTRF2 overexpression were inoculated into backs of immunodeficient Nude mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) NK recruitment , degranulation MDSC infiltration were monitored by FACS Density plot of CD11bHi GR1Hi MDSC in control or anti - GR1 - treated Matrigel plugs ( K ) .

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BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) and NK cells recruitment, degranulation and MDSC infiltration were monitored by FACS Density plot of CD11bHi GR1Hi MDSC staining in control or anti-GR1-treated Matrigel plugs (K).


BJcl2 with or without hTRF2 overexpression were inoculated into backs of immunodeficient Nude mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) NK recruitment , degranulation MDSC infiltration were monitored by FACS Box plots of percentages of infiltrating or activated NK ( CD107a or CD69 ) ( L ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

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BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) and NK cells recruitment, degranulation and MDSC infiltration were monitored by FACS Box plots of the percentages of infiltrating or activated NK cells (CD107a or CD69) (L) Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


BJcl2 with or without hTRF2 overexpression were inoculated into backs of immunodeficient Nude mice treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) NK recruitment , degranulation MDSC infiltration were monitored by FACS Box plots of percentages of CD11bHi GR1Hi MDSC or NKp46 + NK ( M ) infiltrating Matrigel plugs among CD45 + . Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or indicated parent population or number of lung nodules . experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Mann - Whitney .

BJcl2 cells with or without hTRF2 overexpression were inoculated into the backs of immunodeficient Nude mice and treated with anti-GR1 antibody or isotypic control (200µg/IP) and NK cells recruitment, degranulation and MDSC infiltration were monitored by FACS Box plots of the percentages of CD11bHi GR1Hi MDSC or NKp46+ NK cells (M) infiltrating the Matrigel plugs assay among the CD45+ cells. Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


of STAT3 phosphorylation by MSC2 after 18 hours ' co - culture with BJcl2 overexpressing or compromised for TRF2 Histograms of pSTAT3 mean ( MFI ) ( B ) .

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Analysis of STAT3 phosphorylation by MSC2 cells after 18 hours' co-culture with BJcl2 overexpressing or compromised for TRF2 Histograms of the pSTAT3 mean fluorescence intensity (MFI) (B).


+ / - inhibitors : anti - MyD88 peptide inhibitor , anti - mouse TLR2 antibody , or lipopolysaccharide ( LPS ) - RS , an antagonist of TLR4 in ( D )

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+/- inhibitors: anti-MyD88 peptide inhibitor, anti-mouse TLR2 antibody, or lipopolysaccharide (LPS)-RS, an antagonist of TLR4 in (D)


+ / - inhibitors : anti - mouse IL - 6 or JAK1 / 2 inhibitor ( Ruxolitinib ) in ( E ) .

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+/- inhibitors: anti-mouse IL-6 or JAK1/2 inhibitor (Ruxolitinib) in (E).


TRF2 - overexpressing or - knockdown BJcl2 were co - cultured with MSC2 for 18h + / - mouse IL6 blocking antibody or Ruxolitinib . After FACS sorting , MSC2 were co - cultured for 18h with primed purified NK before a 4h challenge with YAK - 1 . NK degranulation IFN - γ production were analyzed by FACS ( G ) . Data information : Panels show points with mean ± corresponding to percentages of each population among NK ( G ) ( n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - ) .

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TRF2-overexpressing or -knockdown BJcl2 cells were co-cultured with MSC2 cells for 18h +/- mouse IL6 blocking antibody or Ruxolitinib. After FACS sorting, MSC2 cells were co-cultured for 18h with primed and purified NK cells before a 4h challenge with YAK-1. NK cell degranulation and IFN-γ production were analyzed by FACS (G). Data information: Panels show all points with mean ± SEM corresponding to the percentages of each cell population among NK cells (G) (n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001; Student's t-test).


TRF2 - overexpressing or - knockdown BJcl2 were co - cultured with MSC2 for 18h + / - mouse IL6 blocking antibody or Ruxolitinib . Alternatively , a 4h challenge with NIH3T3 was performed ability of NK to kill NIH3T3 were determined by colorimetric ( AlamarBlue ) ( H ) . Data information : Panels show points with mean ± corresponding to of specific target lysis ( H ) ( n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - ) .

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TRF2-overexpressing or -knockdown BJcl2 cells were co-cultured with MSC2 cells for 18h +/- mouse IL6 blocking antibody or Ruxolitinib. Alternatively, a 4h challenge with NIH3T3 cells was performed and the ability of NK cells to kill NIH3T3 cells were determined by colorimetric analysis (AlamarBlue) (H). Data information: Panels show all points with mean ± SEM corresponding to the percentage of specific target lysis (H) (n = 3 independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001; Student's t-test).


Gene expression by quantitative polymerase chain reaction ( qPCR ) of TRF2 target genes following TRF2 overexpression or knockdown . Data information : data represent n = 3 independent experiments with mean ±

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Gene expression analysis by quantitative polymerase chain reaction (qPCR) of TRF2 target genes following TRF2 overexpression or knockdown. Data information: data represent n = 3 independent experiments with mean ± SEM


B , C of H3K27ac ( B ) or H3K23me3 ( C ) peaks on 4 kilobase ( Kb ) region around genomic ITSs ( ENCODE data for K562 ) . mean of region is depicted in histograms . ITSs are clustered depending of peak mean obtained from H3K27ac or H3K27me3 immunoprecipitation sequencing ( ChIP - seq ) of 4 Kb region . ITSs detected by ChIP - seq using an anti - TRF2 antibody are shown on left .

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B, C Heatmap of H3K27ac (B) or H3K23me3 (C) peaks on the 4 kilobase (Kb) region around genomic ITSs (ENCODE data for the K562 cell line). The mean intensity of the region is depicted in the histograms. ITSs are clustered depending of the peak mean intensity obtained from H3K27ac or H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) of the 4 Kb region. ITSs detected by ChIP-seq using an anti-TRF2 antibody are shown on the left.


D Specific qPCR for HS3SH4 , GPC6 , VCAN ITSs after ChIP using an anti - H3K27ac antibody in TRF2 - overexpressing or - knockdown BJcl2 . Data represent fold enrichment of H3K27ac level compared with total H3 level . Data information : data represent n = 3 independent experiments with mean ±

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D Specific qPCR for HS3SH4, GPC6, and VCAN ITSs after ChIP using an anti-H3K27ac antibody in TRF2-overexpressing or -knockdown BJcl2 cells. Data represent fold enrichment of the H3K27ac level compared with the total H3 level. Data information: data represent n = 3 independent experiments with mean ± SEM


B - E of CD11bHi GR1Hi MDSCs ( B ) , CD3 - NKp46 + NK ( C ) , NKp46 + CD107a + NK ( D ) or CD69 + NK ( E ) Data information : data are presented by Box plots Min to Max showing points with median corresponding to percentages of each population among CD45 + or NK ( n = 8 mice per group ; * p < 0 . 05 * * * p < 0 . 001 ; Mann - Whitney ) .

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B-E Percentage of CD11bHi GR1Hi MDSCs (B), CD3- NKp46+ NK cells (C), NKp46+ CD107a+ NK cells (D) or CD69+ NK cells (E) Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ or NK cells (n = 8 mice per group; *p < 0.05 and ***p < 0.001; Mann-Whitney test).


Histograms of pSTAT3 mean ( MFI ) of MSC2 when TRF2 was overexpressed target gene knocked down ( G ) Data information MFI of n = 3 independent experiment is normalized to controls represented as fold change of MFI ( n = 3 independent experiments ; * p < 0 . 05 * * p < 0 . 01 ; Student ' s t - ) .

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Histograms of the pSTAT3 mean fluorescence intensity (MFI) of MSC2 cells when TRF2 was overexpressed and the target gene knocked down (G) Data information MFI of n=3 independent experiment is normalized to controls and represented as fold change of MFI (n = 3 independent experiments; *p < 0.05 and **p < 0.01; Student's t-test).


Histograms of pSTAT3 mean ( MFI ) of MSC2 when both TRF2 its target gene were knocked down ( H ) . Data information : MFI of n = 3 independent experiment is normalized to controls represented as fold change of MFI ( n = 3 independent experiments ; * p < 0 . 05 * * p < 0 . 01 ; Student ' s t - ) .

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Histograms of the pSTAT3 mean fluorescence intensity (MFI) of MSC2 cells when both TRF2 and its target gene were knocked down (H). Data information: MFI of n=3 independent experiment is normalized to controls and represented as fold change of MFI (n = 3 independent experiments; *p < 0.05 and **p < 0.01; Student's t-test).


I Histograms of weights on day 19 after implantation of subcutaneous xenografts containing BJcl2 overexpressing or knocked down for HS3ST4 or GPC6 Data information : data are represented as mean + / - ( n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 ; Mann - Whitney ) .

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I Histograms of tumor weights on day 19 after implantation of subcutaneous xenografts containing BJcl2 cells overexpressing or knocked down for HS3ST4 or GPC6 Data information: data are represented as mean +/- SEM (n = 8 mice per group; *p < 0.05, and **p < 0.01; Mann-Whitney test).


A of Heparan Sulfate ( HS ) expression by FACS using 10E4 antibody . Data information histograms representative ( left panels ) MFIs from three independent experiments ( * p < 0 . 05 * * p < 0 . 01 ; Student ' s t - ) are shown + / - .

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A Analysis of Heparan Sulfate (HS) expression by FACS using the 10E4 antibody. Data information histograms representative (left panels) and MFIs from three independent experiments (*p < 0.05 and **p < 0.01; Student's t-test) are shown +/- SEM.


B Atomic Force Microscopic ( AFM ) of glycocalyx stiffness glycocalyx . Data information : In ( B ) , Box plots Min to Max showing points with median represent four independent experiments , each performed using at least 20 ( * p < 0 . 05 ; Mann - Whitney ) .

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B Atomic Force Microscopic (AFM) analysis of glycocalyx and cell stiffness and glycocalyx length. Data information: In (B), Box plots Min to Max showing all points with median represent four independent experiments, each performed using at least 20 cells (*p< 0.05; Mann-Whitney test).


C AFM showing Modulus ( stiffness ) in GFP - expressing or TRF2 - overexpressing cancer . At each of 128x128 force curve points , corresponding to an of 10 μm2 , AFM software automatically displays Sneddon modulus while scanning sample . color bar on left of shows a qualitative representation of elastic map .

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C AFM images showing the Modulus heatmap (stiffness) in GFP-expressing or TRF2-overexpressing cancer cells. At each of the 128x128 force curve points, corresponding to an area of 10 μm2, the AFM software automatically displays the Sneddon modulus analysis while scanning the sample. The color bar on the left of the images shows a qualitative representation of the elastic map.


D of pSTAT3 in MDSCs after co - culture with GFP - expressing or TRF2 - overexpressing cancer treated with or without sodium chlorate ( NaClO3 ) . Data information : In ( D ) , data represents mean of n = 3 independent experiments + / - ( * * * p < 0 . 001 ; Student ' s t - ) .

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D Analysis of pSTAT3 levels in MDSCs after co-culture with GFP-expressing or TRF2-overexpressing cancer cells treated with or without sodium chlorate (NaClO3). Data information: In (D), data represents mean of n = 3 independent experiments +/- SEM (***p < 0.001; Student's t-test).


A Kaplan - Meier curves for overall survival of chemotherapy - treated according to TRF2 expression level Data information : optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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A Kaplan-Meier curves for overall survival of chemotherapy-treated patients according to the TRF2 expression level Data information: the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.


Immunocompetent mice were subcutaneously engrafted with B16F10 with or without TRF2 overexpression treated with three intraperitoneal injections of 50 mg / kg 5 - FU . volume was determined every 2 days using a caliper , growth curves over time ( C ) Data information : , data represent mean values + / - ( n = 8 mice per group ; * p < 0 . 05 * * p < 0 . 01 ; Mann - Whitney ) .

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Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. The tumor volume was determined every 2 days using a caliper, and growth curves over time (C) Data information: , data represent mean values +/- SEM (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test).


Immunocompetent mice were subcutaneously engrafted with B16F10 with or without TRF2 overexpression treated with three intraperitoneal injections of 50 mg / kg 5 - FU . volume were determined on day 19 represented using Box plots Min to Max showing points with median ( D ) . Data information : ( D ) , data are represented by Box plots Min to Max showing points with median ( n = 8 mice per group ; * p < 0 . 05 * * p < 0 . 01 ; Mann - Whitney )

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Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. tumor volume were determined on day 19 and represented using Box plots Min to Max showing all points with median (D). Data information: (D), data are represented by Box plots Min to Max showing all points with median (n = 8 mice per group; *p < 0.05 and **p < 0.01; Mann-Whitney test)


Immunocompetent mice were subcutaneously engrafted with B16F10 with or without TRF2 overexpression treated with three intraperitoneal injections of 50 mg / kg 5 - FU . Overall survival was also determined ( E ) .

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Immunocompetent mice were subcutaneously engrafted with B16F10 cells with or without TRF2 overexpression and treated with three intraperitoneal injections of 50 mg/kg 5-FU. Overall survival was also determined (E).


A Kaplan - Meier curves for overall survival of at stages of cancers ( breast , gastric , ovarian , lung cancer ) depending on TRF2 expression level . Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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A Kaplan-Meier curves for overall survival of patients at all stages of cancers (breast, gastric, ovarian, and lung cancer) depending on TRF2 expression level. Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.


overall survival of gastric cancer ( stages ) was analyzed depending on TRF2 its targets genes expression level ( HS3ST4 , GPC6 VCAN collectively named HSPG genes ) Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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The overall survival of gastric cancer patients (all stages) was analyzed depending on TRF2 and its targets genes expression level (HS3ST4, GPC6 and VCAN collectively named the HSPG genes) Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.


overall survival of gastric cancer ( stages ) was analyzed depending on TRF2 with MDSC level ( Lo or Hi ) , determine as mean expression level of CD33 C5AR , using KMplot Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

The overall survival of gastric cancer patients (all stages) was analyzed depending on TRF2 and with MDSC level (Lo or Hi), determine as the mean expression level of CD33 and C5AR, using KMplot Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.


E Repartition of number of gastric cancer depending on TRF2 MDSC classification . Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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E Repartition of number of gastric cancer patients depending on TRF2 and MDSC classification. Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.


F Overall survival of gastric cancer ( stages ) analyzed depending on TRF2 , HSPG genes ( HS3ST4 , GPC6 VCAN ) MDSC level ( Lo or Hi ) . Data information : For panels , optimal cut - off is determined on KMplot . p value ( log Rank ) , Hazard number of are indicated .

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F Overall survival of gastric cancer patients (all stages) analyzed depending on TRF2, HSPG genes (HS3ST4, GPC6 and VCAN) and MDSC level (Lo or Hi). Data information: For all panels, the optimal cut-off is determined on KMplot. The p value (log Rank test), the Hazard Ratio and number of patients are indicated.