Figure 2B , C Colocalization of N1 - 54 : mVenus : OsCRT3 with OFP - HDEL . OFP - HDEL is an ER lumen marker harboring a C - terminal HDEL ER - retention sequence . ( C ) shows a magnification of the box represented in ( B ) . Bar : 20 μm . D Colocalization analyses along the white arrow in ( C ) . E , F Colocalization of N1 - 54 : mVenus : OsCRT3 with the ER membrane marker AtCNX1 - mCherry . ( F ) shows a magnification of the box represented in ( E ) . Bar : 20 μm . G Colocalization analyses along the white arrow in ( F ) .

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Figure 2B, C Colocalization of N1-54:mVenus:OsCRT3 with OFP-HDEL. OFP-HDEL is an ER lumen marker harboring a C-terminal HDEL ER-retention sequence. (C) shows a magnification of the box represented in (B). Bar: 20 μm. D Colocalization analyses along the white arrow in (C).E, F Colocalization of N1-54:mVenus:OsCRT3 with the ER membrane marker AtCNX1-mCherry. (F) shows a magnification of the box represented in (E). Bar: 20 μm. G Colocalization analyses along the white arrow in (F).


Figure 3C Cold response of cytosolic free Ca2 + concentration in root cells expressing NES - YC3 . 6 . Superimposition of representative normalized ratio variations for wild - type , oscrt3 - 1 , and the complemented transgenic pOsCRT3 root cells . The rectangle represents the regions of interest ( ROIs ) for the ratio measurements . The blue background indicates duration of cold treatment . Bar : 50 μm .

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Figure 3C Cold response of cytosolic free Ca2+ concentration in root cells expressing NES-YC3.6. Superimposition of representative normalized ratio variations for wild-type, oscrt3-1, and the complemented transgenic pOsCRT3 root cells. The rectangle represents the regions of interest (ROIs) for the ratio measurements. The blue background indicates duration of cold treatment. Bar: 50 μm.


Figure 4B Yeast two - hybrid analysis to test the OsCRT3 - OsCIPK7 interaction . The interactions between OsCRT3 , OsCRT3N , OsCRT3P or OsCRT3C with OsCIPK7 , OsCIPK7N , OsCIPK7C , OsCIPK7NAF , or OsCIPK7PPI were verified . C Pull - down assay of the interaction between OsCRT3 and OsCIPK7 . E Localization of OsCIPK7 - GFP in N . benthamiana leaf epidermal cells . OFP - HDEL was used as an ER marker . Bar : 20 μm . F Magnification of the box represented in ( E ) . Colocalization analyses along the white arrow are shown . Bar : 20 μm .

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Figure 4B Yeast two-hybrid analysis to test the OsCRT3-OsCIPK7 interaction. The interactions between OsCRT3, OsCRT3N, OsCRT3P or OsCRT3C with OsCIPK7, OsCIPK7N, OsCIPK7C, OsCIPK7NAF, or OsCIPK7PPI were verified.C Pull-down assay of the interaction between OsCRT3 and OsCIPK7.E Localization of OsCIPK7-GFP in N. benthamiana leaf epidermal cells. OFP-HDEL was used as an ER marker. Bar: 20 μm. F Magnification of the box represented in (E). Colocalization analyses along the white arrow are shown. Bar: 20 μm.


Figure 5A OsCIPK7 interacts with OsCBLs in a yeast two - hybrid system . Interactions were verified by growing the yeast on selective medium ( SD / - Leu - Trp , SD / - Leu - Trp - His - Ade ) and conducting β - Galactosidase assays . B Subcellular localization of OsCBL7 and OsCBL8 in N . benthamiana leaf epidermal cells . Pictures were taken after 2 and 3 days of expression . Bar : 50 μm . C Bimolecular fluorescence complementation ( BiFC ) assay confirming the interaction between OsCBL7 / 8 and OsCIPK7 in N . benthamiana leaf epidermal cells . 19K was used as a negative control . Bar : 50 μm . D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three - hybrid assay . E Constructs and OsCRT3 protein expression level in ( D ) . 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 the expression of OsCIPK7 , OsCBL7 , OsCBL8 , and OsCRT3 in ( F ) respectively . 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 .

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Figure 5A OsCIPK7 interacts with OsCBLs in a yeast two-hybrid system. Interactions were verified by growing the yeast on selective medium (SD/-Leu-Trp, SD/-Leu-Trp-His-Ade) and conducting β-Galactosidase assays.B Subcellular localization of OsCBL7 and OsCBL8 in N. benthamiana leaf epidermal cells. Pictures were taken after 2 and 3 days of expression. Bar: 50 μm.C Bimolecular fluorescence complementation (BiFC) assay confirming the interaction between OsCBL7/8 and OsCIPK7 in N. benthamiana leaf epidermal cells. 19K was used as a negative control. Bar: 50 μm.D Interactions between OsCIPK7 and OsCBL7 or OsCBL8 were attenuated by OsCRT3 in a yeast three-hybrid assay.E Constructs and OsCRT3 protein expression level in (D).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 the expression of OsCIPK7, OsCBL7, OsCBL8, and OsCRT3 in (F) respectively.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.


Figure 7A 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 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 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 .

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Figure 7A 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 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 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.


figf1 ( 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 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 ) 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 ) 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 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 ) 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 ) .

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figf1(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 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) 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) 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 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) 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).


figf2 ( 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 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 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 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 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 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 .

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figf2(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 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 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 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 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 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.


figf3 ( 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 ) 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 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 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 .

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figf3(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) 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 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 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.


figf4 ( 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 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 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 ) 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 .

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

figf4(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 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 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) 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.


figf5 ( 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 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 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 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 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 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 .

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

figf5(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 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 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 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 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 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.


figf6 ( 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 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 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 .

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

figf6(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 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 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.


figf7 ( 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 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 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 .

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

figf7(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 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 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.


figf8 ( 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 .

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

figf8(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.


Figure 1A 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 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 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 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 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 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 .

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

Figure 1A 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 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 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 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 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 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.


Figure 2A 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 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 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 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 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 ) .

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

Figure 2A 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 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 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 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 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).


Figure 3A 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 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 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 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 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 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 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 ) .

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

Figure 3A 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 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 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 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 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 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 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).


Figure 4A - 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 ) .

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Figure 4A-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).


Figure 5A 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 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 ) .

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Figure 5A 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 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).


Figure 6A 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 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 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 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 ) .

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Figure 6A 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 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 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 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).


Figure 7A , 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 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 ) .

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

Figure 7A, 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 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).


Figure 8Human 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 - 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 - 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 - 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 stem cells ( iPSCs ) derived from a FXS patient ( FXS - iPSC ) and the isogenic rescue cells ( C1 _ 2 - iPSC ) were analysed by immunofluorescence microscopyHuman 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 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 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 ) .

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

Figure 8Human 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-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-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-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 stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopyHuman 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 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 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).


Figure 9A 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 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 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 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 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 (

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

Figure 9A 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 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 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 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 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 (


Figure 1A 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 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 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 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 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 . 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 .

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

Figure 1A 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 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 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 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 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.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.


Figure 2B16F10 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 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 . 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 . 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 . 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 . 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 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 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 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 .

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

Figure 2B16F10 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 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.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.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.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.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 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 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 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.


Figure 3Analysis 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 ) + / - inhibitors : anti - mouse IL - 6 or JAK1 / 2 inhibitor ( Ruxolitinib ) in ( E ) . 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 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 ) .

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

Figure 3Analysis 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)+/- inhibitors: anti-mouse IL-6 or JAK1/2 inhibitor (Ruxolitinib) in (E).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 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).