( H ) Larval brains of control ( ctrl ; yw ) , cdk1 - Myc overexpression under insc - Gal4 , pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) , pr - set7716 / Df with cdk1 - Myc induced with insc - Gal4 at 24h ALH were labelled with Dpn EdU . ( I , J ) Quantifications of EdU incorporation ( I ) NSC diameters ( J ) for genotypes in ( H ) . In ( I ) , n = 1334 , 16 BL for control ; n = 1299 , 15 BL for insc > cdk1 ; n = 1221 , 18 BL for pr - set7716 / Df n = 1372 , 16 BL for pr - set7716 / Df with cdk1 - Mycinduced with insc - Gal4 . In ( J ) , n = 153 for control ; n = 152 for insc > cdk1 ; n = 156 for pr - set7716 / Df n = 159 for pr - set7716 / Df with cdk1 - Myc induced with insc - Gal4 . Data information : Data were presented in graphs as mean ± SD I , J ) . n numbers in I ) were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing among different genotypes using ordinary one - way ANOVA * * * P < 0 . 001 . Scale bars , 30 μm H ) . Control : insc - Gal4 / + UAS -

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

(H) Larval brains of control (ctrl; yw), cdk1-Myc overexpression under insc-Gal4, pr-set7716/Df(3R)ED5644 (716/Df), and pr-set7716/Df with cdk1-Myc induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU. (I, J) Quantifications of EdU incorporation (I) and NSC diameters (J) for genotypes in (H). In (I), n=1334, 16 BL for control; n=1299, 15 BL for insc>cdk1; n=1221, 18 BL for pr-set7716/Df and n=1372, 16 BL for pr-set7716/Df with cdk1-Mycinduced with insc-Gal4. In (J), n=153 for control; n=152 for insc>cdk1; n=156 for pr-set7716/Df and n=159 for pr-set7716/Df with cdk1-Myc induced with insc-Gal4.Data information: Data were presented in quantification graphs as mean ± SD I, J). n numbers in I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA ***P < 0.001. Scale bars, 30 μm H). Control: insc-Gal4/+ UAS-dicer2


( A ) Larval NSCs of control ( ctrl ; yw ) ebd1240 at 24h ALH were stained for Deadpan ( Dpn ) Miranda ( Mira ) . ( B ) Quantifications of extensions for genotypes in ( A ) . Control : n = 886 , 11 BL ; ebd1240 : n = 864 , 12 BL . ( C ) Larval brains of control ( ctrl ; yw ) ebd1240 at 24h ALH were labelled with Dpn EdU . ( D ) Quantifications of NSC without EdU incorporation for genotypes in ( C ) . Control : n = 1034 , 12 BL ; ebd1240 : n = 1139 , 16 BL . Data information : Data are presented in ( B , D as mean ± SD . n numbers in ( B , D , were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing two different genotypes using Mann - Whitney ( B , D ns : non - significant ; * P < 0 . 05 ; * * * P < 0 . 001 .

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

(A) Larval NSCs of control (ctrl; yw) and ebd1240 at 24h ALH were stained for Deadpan (Dpn) and Miranda (Mira). (B) Quantifications of cellular extensions for genotypes in (A). Control: n=886, 11 BL; ebd1240: n=864, 12 BL. (C) Larval brains of control (ctrl; yw) and ebd1240 at 24h ALH were labelled with Dpn and EdU. (D) Quantifications of NSC without EdU incorporation for genotypes in (C). Control: n=1034, 12 BL; ebd1240: n=1139, 16 BL. Data information: Data are presented in (B, D as mean ± SD. n numbers in (B, D, were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (B, D ns: non-significant; *P < 0.05; ***P < 0.001.


( H ) Larval brains of control ( ctrl ; yw ) , ebd1 - Flag overexpression under insc - Gal4 , pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) pr - set7716 / Df with ebd1 induced with insc - Gal4 at 24h ALH were labelled with Dpn EdU . ( I ) Quantifications of NSCs without EdU incorporation for larval brains in ( H ) . Control : n = 1162 , 13 BL ; insc > ebd1 : n = 997 , 11 BL ; pr - set7716 / Df : n = 928 , 13 BL pr - set7716 / Df with ebd1 induced with insc - Gal4 : n = 1284 , 16 BL . Data information : Data are presented in I ) as mean ± SD . n numbers in I ) were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing among different genotypes using ordinary one - way ANOVA I ) . ns : non - significant ; * P < 0 . 05 ; * * * P < 0 . 001 .

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

(H) Larval brains of control (ctrl; yw), ebd1-Flag overexpression under insc-Gal4, pr-set7716/Df(3R)ED5644 (716/Df) and pr-set7716/Df with ebd1 induced with insc-Gal4 at 24h ALH were labelled with Dpn and EdU. (I) Quantifications of NSCs without EdU incorporation for larval brains in (H). Control: n=1162, 13 BL; insc>ebd1: n=997, 11 BL; pr-set7716/Df: n=928, 13 BL and pr-set7716/Df with ebd1 induced with insc-Gal4: n=1284, 16 BL. Data information: Data are presented in I) as mean ± SD. n numbers in I) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing among different genotypes using ordinary one-way ANOVA I). ns: non-significant; *P < 0.05; ***P < 0.001.


( A ) Larval NSCs of control ( ctrl ; yw ) cdk1B47 at 24h ALH were labelled for Earthbound1 ( Ebd1 ) , Deadpan ( Dpn ) , Miranda ( Mira ) . ( B ) Quantifications of corrected total ( CCF ) of Ebd1 in genotypes in ( A ) . CCF = Integrated Density - ( of selected × Mean of background readings ) . CCFs were 846 . 9 ± 394 . 2 for control ( ctrl ; yw ; n = 167 , 14 BL ) 354 . 3 ± 245 . 3 for cdk1B47 ( n = 211 , 13 BL ) . ( C , D ) Larval NSCs of control ( UAS - dicer2 ) UAS - cdk1 - Myc induced with insc - Gal4 at 6h ALH were labelled for Ebd1 , Dpn , Mira . ( D ) Quantifications of CCF of Ebd1 in genotypes in ( C ) . CCFs were 111 . 3 ± 80 . 2 for control ( n = 157 , 12 BL ) 346 . 6 ± 221 . 1 for cdk1 - Myc ( n = 181 , 12 BL ) . Data information : Data are presented in ( B , D , as mean ± SD . n numbers in ( B , D , were total NSCs counted with total brain lobes measured , while each NSC was considered as one biological replicate . arrows point to NSCs ( A , C , Yellow dotted boxes indicate region of zoomed - in ( A , C , Statistical analyses were done comparing between two different genotypes using Mann - Whitney ( B , D ) Scale bars , 15 μm ( A , C ,

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

(A) Larval NSCs of control (ctrl; yw) and cdk1B47 at 24h ALH were labelled for Earthbound1 (Ebd1), Deadpan (Dpn), and Miranda (Mira). (B) Quantifications of the corrected total cell fluorescence (CCF) of Ebd1 in genotypes in (A). CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 846.9±394.2 for control (ctrl; yw; n=167, 14 BL) and 354.3±245.3 for cdk1B47 (n=211, 13 BL). (C, D) Larval NSCs of control (UAS-dicer2) and UAS-cdk1-Myc induced with insc-Gal4 at 6h ALH were labelled for Ebd1, Dpn, and Mira. (D) Quantifications of the CCF of Ebd1 in genotypes in (C). CCFs were 111.3±80.2 for control (n=157, 12 BL) and 346.6±221.1 for cdk1-Myc (n=181, 12 BL). Data information: Data are presented in (B, D, as mean ± SD. n numbers in (B, D, were total NSCs counted with total brain lobes measured, while each NSC was considered as one biological replicate. White arrows point to NSCs (A, C, Yellow dotted boxes indicate the region of zoomed-in images (A, C, Statistical analyses were done comparing between two different genotypes using Mann-Whitney test (B, D) Scale bars, 15 μm (A, C,


( E ) qPCR of cdk1 in larval brains of control ( ctrl ; yw ) ebd1240 at 24h ALH . n = 3 as biological replicates . Statistical analyses were done comparing control ( GAPDH1 , GAPDH2 , Tbp1 Rpl32 ) ebd1240 , using two - Student ' s t - . mRNA of control were normalized to 1 . Data information : Statistical analyses were done comparing between two different genotypes using two - Student ' s t - ( E ) .

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

(E) qPCR analysis of cdk1 in larval brains of control (ctrl; yw) and ebd1240 at 24h ALH. n=3 as biological replicates. Statistical analyses were done comparing control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and ebd1240, using two-tailed Student's t-test. The mRNA levels of control were normalized to 1. Data information: Statistical analyses were done comparing between two different genotypes using two-tailed Student's t-test (E).


Larval NSCs of control ( ctrl ; yw ) , ebd1240 pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) at 24h ALH were stained for Senseless ( Sens ) , Dpn Mira . ( F ) Data information : arrows point to NSCs F ) . Yellow dotted boxes indicate region of zoomed - in F ) . Scale bars , 15 μm F ) .

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

Larval NSCs of control (ctrl; yw), ebd1240 and pr-set7716/Df(3R)ED5644 (716/Df) at 24h ALH were stained for Senseless (Sens), Dpn and Mira. (F) Data information: White arrows point to NSCs F). Yellow dotted boxes indicate the region of zoomed-in images F). Scale bars, 15 μm F).


( B ) Representative IF in CHME3 stably expressing Flag alone control or Flag - tagged EB1 constructs depicted in A co - stained for Tyrosinated - MTs ( Tyr - MTs ) , nucleus ( Hoechst ) . Scale bar , 10 μm .

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

(B) Representative IF images in CHME3 stably expressing Flag alone control or the Flag-tagged EB1 constructs depicted in A co-stained for Tyrosinated-MTs (Tyr-MTs), and the nucleus (Hoechst). Scale bar, 10 μm.


( C ) HIV - 1 - VSV - luc infection is enhanced in CHME3 depleted of endogenous EB1 stably expressing full - ( FL ) EB1 , but not in control Flag or any of other EB1 constructs depicted in A .

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

(C) HIV-1-VSV-luc infection is enhanced in CHME3 depleted of endogenous EB1 and stably expressing full-length (FL) EB1, but not in control Flag or any of the other EB1 constructs depicted in A.


( D ) Increasing amount of FL - EB1 , but not ∆ EEY results in a dose - dependent enhancement of HIV - 1 - VSV - luc infection in transiently transfected CHME3 .

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

(D) Increasing amount of FL-EB1, but not ∆EEY results in a dose-dependent enhancement of HIV-1-VSV-luc infection in transiently transfected CHME3 cells.


HIV - 1 - VSV - luc ( E ) infection is reduced in differentiated THP - 1 depleted of CLIP170 determined by luciferase assays or FACS , respectively .

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

HIV-1-VSV-luc (E) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively.


HIV - 1 - VSV - ZsGreen at various dilutions ( corresponding to multiplicities of infection ( m . o . i ) 0 . 06 , 0 . 03 0 . 02 ) ( F ) infection is reduced in differentiated THP - 1 depleted of CLIP170 determined by luciferase assays or FACS , respectively .

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

HIV-1-VSV-ZsGreen at various dilutions (corresponding to multiplicities of infection (m.o.i) 0.06, 0.03 and 0.02) (F) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively.


( G ) HIV - 1 - WT - luc infection is decreased in CLIP170 depleted CHME3 .

(G) HIV-1-WT-luc infection is decreased in CLIP170 depleted CHME3 cells.


Effects of CLIP170 on fusion of viral particles by endocytosis . CHME3 either treated with control or CLIP170 siRNAs ( A ) were infected with Mock or HIV - 1 - VSV - BlaM - Vpr . Representative FACS of ( n = 3 ) showing ∼ 9 % ( Control siRNA ) 3 % ( CLIP170 siRNA ) shift ( A ) from green ( uncleaved CCF2 ) to blue ( CCF2 ) . % infected is indicated at top left corner of each plot from independent repeats is presented as bar graphs at right side of each FACS plot .

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

Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells either treated with control or CLIP170 siRNAs (A) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing ∼ 9% (Control siRNA) and 3% (CLIP170 siRNA) shift (A) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.


HIV - 1 - VSV - luc infection is enhanced in Flag - CLIP170 , but not in Flag control , expressing CHME3 .

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

HIV-1-VSV-luc infection is enhanced in Flag-CLIP170, but not in Flag control, expressing CHME3 cells.


Molecular markers ( in kDa ) are shown to right of WBs in C .

Molecular weight markers (in kDa) are shown to the right of WBs in C.


Effects of CLIP170 on fusion of viral particles by endocytosis . CHME3 expressing Flag control or Flag - CLIP170 ( D ) were infected with Mock or HIV - 1 - VSV - BlaM - Vpr . Representative FACS of ( n = 3 ) showing no difference ( D ) from green ( uncleaved CCF2 ) to blue ( CCF2 ) . % infected is indicated at top left corner of each plot from independent repeats is presented as bar graphs at right side of each FACS plot .

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

Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells expressing Flag control or Flag-CLIP170 (D) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing no difference (D) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.


( E ) fused double - labelled ( GFP - Vpr + / S15 - tomato - ) HIV - 1 - DL - WT are not affected in CHME3 depleted of CLIP170 . Data represent mean of ≥ 895 particles analyzed in ≥ 11 fields of view ( ≥ 17 ) for each condition . One - way ANOVA was used to calculate statistical significance ( mean ± ) .

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

(E) The percentage fused double-labelled (GFP-Vpr+/S15-tomato-) HIV-1-DL-WT are not affected in CHME3 cells depleted of CLIP170. Data represent mean of ≥895 virus particles analyzed in ≥11 fields of view (≥17 cells) for each condition. One-way ANOVA was used to calculate statistical significance (mean ±SEM).


( F ) HIV - 1 - WT - GFP - Vpr particles within 2 μm of nucleus is reduced in CHME3 depleted of CLIP170 at 2 4 h . p . i , scatterplot with mean of ≥ 354 particles quantified in ≥ 13 per sample . Statistical significance was determined by one - way ANOVA * = p < 0 . 05 , * * * = p < 0 . 001 .

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

(F) Percentage HIV-1-WT-GFP-Vpr particles within 2 μm of the nucleus is reduced in CHME3 depleted of CLIP170 at 2 and 4 h.p.i, scatterplot with mean of ≥354 virus particles quantified in ≥13 cells per sample. Statistical significance was determined by one-way ANOVA * = p<0.05, *** = p<0.001.


Infected in F analyzed by live imaging . Scatterplots representing displacement over time ( velocity ) Each dot represents one viral particle tracked , red indicates mean . 188 trajectories were analyzed from 25 movies . Statistical significance was determined by t . * * * = p < 0 . 001 .

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

Infected cells in F analyzed by live cell imaging. Scatterplots representing displacement over time (velocity) Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001.


Infected in F analyzed by live imaging . cumulative travelled over time ( speed ) ( H ) . Each dot represents one viral particle tracked , red indicates mean . 188 trajectories were analyzed from 25 movies . Statistical significance was determined by t . * * * = p < 0 . 001 .

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

Infected cells in F analyzed by live cell imaging. cumulative distance travelled over time (speed) (H). Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001.


( I ) induction of Ac - MTs is blocked in HIV - 1 - VSV - luc ( ) infected , but not in uninfected ( NI ) or Mock infected CHME3 depleted of EB1 , but not CLIP170 at 6 h . p . i . Tyrosinated MTs ( Tyr - MTs ) are not affected . Representative fields are shown . Similar results were obtained in three independent experiments . Scale bar , 50 μm .

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

(I) The induction of Ac-MTs is blocked in HIV-1-VSV-luc (Virus) infected, but not in uninfected (NI) or Mock infected CHME3 depleted of EB1, but not CLIP170 at 6 h.p.i. Tyrosinated MTs (Tyr-MTs) are not affected. Representative fields are shown. Similar results were obtained in three independent experiments. Scale bar, 50 μm.


CHME3 treated with negative control , EB1 or CLIP170 siRNAs were either lysed analyzed by WB Molecular markers ( in kDa ) are shown to right of WBs in A .

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

CHME3 treated with negative control, EB1 or CLIP170 siRNAs were either lysed and analyzed by WB Molecular weight markers (in kDa) are shown to the right of WBs in A.


CHME3 treated with negative control , EB1 or CLIP170 siRNAs were infected with GFP - Vpr / S15 - tomato - labeled VSV - G pseudotyped HIV - 1 ( B ) . Scatterplot of maximum p24 of fused viral particles normalized to control mean .

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

CHME3 treated with negative control, EB1 or CLIP170 siRNAs were infected with GFP-Vpr/S15-tomato-labeled VSV-G pseudotyped HIV-1 (B). Scatterplot of maximum p24 intensity of fused viral particles normalized to the control mean.


( C - D ) Amounts of pelleted p24 CA normalized to input CA at 3 h . p . i . with HIV - 1 - VSV - luc in 293A ( C ) or CHME3 ( D ) treated with control , EB1 or CLIP170 siRNAs . Mean + / - from three independent experiments is shown . Statistical significance was determined by one - way ANOVA . Error bars = standard error of mean ( * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 ) .

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

(C-D) Amounts of pelleted p24 CA normalized to the input CA at 3 h.p.i. with HIV-1-VSV-luc in 293A (C) or CHME3 (D) treated with control, EB1 or CLIP170 siRNAs. Mean +/- SEM from three independent experiments is shown. Statistical significance was determined by one-way ANOVA. Error bars = standard error of the mean (* = p-value ≤0.05, ** = p-value ≤0.01, *** = p-value ≤0.001).


Representative ( n = 3 ) WB demonstrating that while GAPDH - HA control do not bind ( A FEZ1 - Flag control pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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

Representative (n=3) WB analysis demonstrating that while GAPDH-HA control do not bind (A FEZ1-Flag control pelleted with in vitro assembled HIV-1 CA-NC complexes.


Representative ( n = 3 ) WB demonstrating that while EB1 - Flag do not bind pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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

Representative (n=3) WB analysis demonstrating that while EB1-Flag do not bind pelleted with in vitro assembled HIV-1 CA-NC complexes.


Representative ( n = 3 ) WB demonstrating that while Flag - CLIP170 pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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

Representative (n=3) WB analysis demonstrating that while Flag-CLIP170 pelleted with in vitro assembled HIV-1 CA-NC complexes.


( D ) of of bound CLIP170 as a of total across experimental replicates ( n = 3 ) from C . Statistical significance was determined by t ( mean ± ) * * * = p < 0 . 001 .

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

(D) Quantification of the levels of bound CLIP170 as a ratio of total protein across experimental replicates (n=3) from C. Statistical significance was determined by t test (mean ±SEM) *** = p<0.001.


( E ) Representative WB ( n = 3 ) showing of HIV - 1 p24 CA in mock fractions separated by sucrose gradient centrifugation . Fractions 1 - 3 contain free p24 not associated with intact cores , while fractions # 8 # 9 harbouring intact HIV - 1 cores .

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

(E) Representative WB analysis (n=3) showing the levels of HIV-1 p24 CA in mock and virus fractions separated by sucrose gradient centrifugation. Fractions 1-3 contain free p24 not associated with the intact cores, while fractions #8 and # 9 harbouring intact HIV-1 cores.


( F ) Flag - CLIP170 , but not Flag control , also binds to fractions containing intact HIV - 1 cores ( # 8 # 9 ) but not with Mock fractions .

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

(F) Flag-CLIP170, but not Flag control, also binds to fractions containing intact HIV-1 cores (Virus #8 and #9) but not with Mock fractions.


( G ) HIV - 1 - WT - luc infection is increased in CHME3 stably expressing Flag - CLIP170 , but not control Flag or Flag - CLIP170ΔCAPgly . Molecular markers ( in kDa ) are shown to right of WBs .

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

(G) HIV-1-WT-luc infection is increased in CHME3 cells stably expressing Flag-CLIP170, but not control Flag or Flag-CLIP170ΔCAPgly. Molecular weight markers (in kDa) are shown to the right of WBs.


( H ) Representative ( n = 3 ) of CHME3 expressing Flag - CLIP170 or Flag - CLIP170ΔCAPgly stained for Flag Tyr - MTs . Scale bar , 10 μm .

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(H) Representative images (n=3) of CHME3 expressing Flag-CLIP170 or Flag-CLIP170ΔCAPgly stained for Flag and Tyr-MTs. Scale bar, 10 μm.


( A ) Super resolution microscopy ( SIM ) imaging of Flag - tagged , cyclophilin A ( CypA - Flag ) , EB1 - Flag , or Flag - CLIP170 bound to in vitro assembled CA - NC complexes , stained with anti - p24 AG3 . 0 ( Red ) , anti - Flag or anti - CLIP170 ( Green ) . Scale bar , 2 μm . Representative from different fields of view are shown .

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(A) Super resolution microscopy (SIM) imaging of Flag-tagged proteins, cyclophilin A (CypA-Flag), EB1-Flag, or Flag-CLIP170 bound to in vitro assembled CA-NC complexes, stained with anti-p24 AG3.0 (Red), anti-Flag or anti-CLIP170 (Green). Scale bar, 2 μm. Representative images from different fields of view are shown.


( B ) WB confirming expression in samples from A C . Molecular markers ( in kDa ) are shown to right of WBs .

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(B) WB analysis confirming protein expression in samples from A and C. Molecular weight markers (in kDa) are shown to the right of WBs.


( C ) ( n = 3 ) of association of each with 97 to 137 CA - NC complexes , using untransfected lysates ( Control ) EB1 - Flag as controls for non - specific background .

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(C) Quantification (n=3) of the association of each protein with 97 to 137 CA-NC complexes, using untransfected lysates (Control) and EB1-Flag as controls for non-specific background staining.


( D ) Lysates from untransfected , CypA - Flag - or Flag - CLIP170 - transfected were subjected to CA - NC binding assays WB .

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(D) Lysates from untransfected, CypA-Flag- or Flag-CLIP170-transfected cells were subjected to CA-NC binding assays and WB analysis.


( E ) of CypA or CLIP170 bound to WT or R18L CA - NC assemblies in samples from D were quantified using Fiji ( n = 4 experimental replicates ; data is presented as a of CypA or CLIP170 in bound versus input for each CA - NC assembly , normalized to WT CA - NC arbitrarily set to 1 ) . Statistical significance was determined by t ( mean ± ) . * * * = p < 0 . 001 .

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(E) Levels of CypA or CLIP170 bound to WT or R18L CA-NC assemblies in samples from D were quantified using Fiji (n = 4 experimental replicates; data is presented as a ratio of CypA or CLIP170 in bound versus input for each CA-NC assembly, normalized to WT CA-NC arbitrarily set to 1). Statistical significance was determined by t test (mean ±SEM). *** = p<0.001.


Anti - HA co - IP showing that GAPDH fused to HIV - 1 MHR ( GAPDH - MHRhiv ) , but not HA - tagged GAPDH ( GAPDH ) binds to Flag - CLIP170 ( C ) in 293T . Results are representative of three experimental replicates .

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Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv), but not HA-tagged GAPDH (GAPDH) binds to Flag-CLIP170 (C) in 293T cells. Results are representative of three experimental replicates.


Anti - HA co - IP showing that GAPDH fused to HIV - 1 MHR ( GAPDH - MHRhiv ) endogenous forms of various human SxIP - containing + TIPs , but not vimentin ( D ) in 293T . Results are representative of three experimental replicates .

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Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv) endogenous forms of various human SxIP-containing +TIPs, but not vimentin (D) in 293T cells. Results are representative of three experimental replicates.


Anti - HA co - IP showing that GAPDH fused to HIV - 1 MHR ( GAPDH - MHRhiv ) or to Flag - tagged SxIP fragment ( Flag - Clasp2M ) ( E ) in 293T . Results are representative of three experimental replicates .

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Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv) or to Flag-tagged SxIP fragment (Flag-Clasp2M) (E) in 293T cells. Results are representative of three experimental replicates.


HIV - 1 - VSV - luc ( ) , but not uninfected ( NI ) or Mock , infection induces formation of Ac - MTs in CHME3 expressing GAPDH - MHRhiv , but not GAPDH , as shown by WB ( F )

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HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH, as shown by WB analysis (F)


HIV - 1 - VSV - luc ( ) , but not uninfected ( NI ) or Mock , infection induces formation of Ac - MTs in CHME3 expressing GAPDH - MHRhiv , but not GAPDH or imaging at 6 h . p . i ( G ) . Scale bar , 15 μm . Representative fields are shown . Results are representative of three experimental replicates .

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

HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH or imaging at 6 h.p.i (G). Scale bar, 15 μm. Representative fields are shown. Results are representative of three experimental replicates.


( H ) GAPDH - MHRhiv expression in CHME3 reduces infection with HIV - 1 - WT - luc . Statistical significance was determined by t . * = p < 0 . 05 . Data are mean values from three independent experiments + / - . Molecular markers ( in kDa ) are shown to right of WBs .

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(H) GAPDH-MHRhiv expression in CHME3 reduces infection with HIV-1-WT-luc. Statistical significance was determined by t test. * = p<0.05. Data are mean values from three independent experiments +/- SEM. Molecular weight markers (in kDa) are shown to the right of WBs.


( C ) Anti - HA co - IP in CHME3 expressing GAPDH - MHRhiv , GAPDH - MHRsiv or GAPDH - MHRmulv showing binding to endogenous CLIP170 . Results are representative of three experimental replicates .

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(C) Anti-HA co-IP in CHME3 expressing GAPDH-MHRhiv, GAPDH-MHRsiv or GAPDH-MHRmulv showing binding to endogenous CLIP170. Results are representative of three experimental replicates.


Anti - HA co - IP in Rhesus macaque FRhK4 ( D ) expressing GAPDH alongside GAPDH -

Anti-HA co-IP in Rhesus macaque FRhK4 (D) cells expressing GAPDH alongside GAPDH-MHRsiv


Anti - HA co - IP in rat fibroblast Rat2 ( E ) expressing GAPDH alongside GAPDH - MHRmulv Anti - EB1 co - IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 . Results are representative of three experimental replicates .

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Anti-HA co-IP in rat fibroblast Rat2 (E) cells expressing GAPDH alongside GAPDH-MHRmulv Anti-EB1 co-IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 cells. Results are representative of three experimental replicates.


FRhK4 infected with SIV - VSV - luc ( F ) stained for Ac - MTs , Tyr - MTs , nucleus ( Hoechst ) at 6 h . p .

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FRhK4 cells infected with SIV-VSV-luc (F) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i


Rat2 infected with MuLV - VSV - luc ( G ) stained for Ac - MTs , Tyr - MTs , nucleus ( Hoechst ) at 6 h . p . i ,

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Rat2 cells infected with MuLV-VSV-luc (G) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i,


FRhK4 infected with SIV - VSV - luc analyzed by

FRhK4 cells infected with SIV-VSV-luc analyzed by WB


Rat2 infected with MuLV - VSV - luc analyzed by

Rat2 cells infected with MuLV-VSV-luc analyzed by WB


Measurements of SIV - VSV - luc infectivity in FRhK4 ( J ) depleted of CLIP170 . Statistical significance was determined by t . * * * = p < 0 . 001 . Data are mean values from two independent experiments + / - .

Measurements of SIV-VSV-luc infectivity in FRhK4 (J) cells depleted of CLIP170. Statistical significance was determined by t test. *** = p<0.001. Data are mean values from two independent experiments +/- SEM.


Measurements of MuLV - VSV - luc infectivity in Rat2 ( K ) depleted of CLIP170 .

Measurements of MuLV-VSV-luc infectivity in Rat2 (K) cells depleted of CLIP170.


( L ) 293T transfected with GAPDH - MHRhiv or R162Q mutant ( GAPDH - MHRhivQ ) were subjected to anti - HA co - IP . Representative WB is shown ( n = 2 ) . Molecular markers ( in kDa ) are shown to right of WBs .

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(L) 293T transfected with GAPDH-MHRhiv or R162Q mutant (GAPDH-MHRhivQ) were subjected to anti-HA co-IP. Representative WB analysis is shown (n = 2). Molecular weight markers (in kDa) are shown to the right of WBs.


A PhosTag - Western blot probed for YAP reveals increased of unphosphorylated ( downshift ) YAP in HEK293A in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure ( + ) compared to control ( - ) . B PhosTag - Western blot probed for TAZ reveals increased of unphosphorylated ( downshift ) TAZ in HEK293A in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure ( + ) compared to control ( - ) .

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A PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-). B PhosTag-based Western blot probed for TAZ reveals increased levels of unphosphorylated (downshift) TAZ in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-).


C Western blots from lysates as in ( A ) ( B ) show total in control ( - ) in lysates obtained from exposed to hydrostatic pressure ( + ) stimulation in HEK293A . Note decrease in inhibitory p127 YAP signal as well as increase of YAP / TAZ targets CTGF CYR61 upon hydrostatic pressure .

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C Western blots from cell lysates as in (A) and (B) show total protein levels in control (-) and in cell lysates obtained from cells exposed to hydrostatic pressure (+) stimulation in HEK293A cells. Note the decrease in the inhibitory p127 YAP signal as well as increase of YAP/TAZ targets CTGF and CYR61 levels upon hydrostatic pressure.


D PhosTag - Western blot probed for YAP reveals increased of unphosphorylated ( downshift ) YAP in 143B in response to hydrostatic pressure ( + ) compared to control ( - ) .

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D PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in 143B cells in response to hydrostatic pressure (+) compared to control (-).


E Western blot shows total in control ( - ) with hydrostatic pressure ( + ) stimulation in 143B from lysates as in D .

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E Western blot shows total protein levels in control (-) and with hydrostatic pressure (+) stimulation in 143B cells from cell lysates as in D.


F HEK293A at steady state ( upper ) or upon cyclic 0 . 1Hz 200mbar hydrostatic pressure ( lower ) were analysed by immunofluorescence . are labelled for Hoechst ( blue ) YAP ( green ) . Scale bar = 20µm . Graph on right depict of - to - ( Nucl / Cyt ) of YAP in as shown on left . Each dot represents a single data are pooled from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U . * * * p < 0 . 001 . G 143B at steady state ( upper ) or upon cyclic 0 . 1Hz 200mbar hydrostatic pressure ( lower ) . Graph on right show of - to - ( Nucl / Cyt ) of YAP in 143B from as those shown on left . Each dot represents a single data are pooled from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U . * * * p < 0 . 001 .

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F HEK293A cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower) were analysed by immunofluorescence. Cells are labelled for Hoechst (blue) and YAP (green). Scale bar = 20µm. Graph on the right depict quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in cells as shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001. G 143B cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower). Graph on the right show quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in 143B from images as those shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001.


H Western blot confirming no YAP TAZ expression in two independent ( # 1 # 2 ) YAP / TAZ double knockout ( Y / T DKO ) clones .

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H Western blot confirming no YAP and TAZ expression in two independent (#1 and #2) YAP/TAZ double knockout (Y/T DKO) clones.


I Relative expression of YAP / TAZ target genes CYR61 CTGF in HEK293A WT Y / T DKO clones in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure . Data from six independent experiments Error bars represent mean ± SD . Kruskal - Wallis with Dunn ' s post hoc . * * p = 0 . 0661 ( WT CYR61 ) , * * * p = 0 . 0001 ( WT CTGF ) , p = 0 . 3924 ( Y / T DKO # 1 CYR61 ) , p = 0 . 4616 ( Y / T DKO # 1 CTGF ) , p > 0 . 999 ( Y / T DKO # 2 CYR61 ) , p = 0 . 0782 ( Y / T DKO # 2 CTGF ) .

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I Relative expression levels of YAP/TAZ target genes CYR61 and CTGF in HEK293A WT and Y/T DKO clones in response to cyclic 0.1Hz 200mbar hydrostatic pressure. Data from six independent experiments Error bars represent mean ± SD. Kruskal-Wallis test with Dunn's post hoc. **p=0.0661 (WT CYR61), ***p= 0.0001 (WT CTGF), p=0.3924 (Y/T DKO#1 CYR61), p=0.4616 (Y/T DKO#1 CTGF), p>0.999 (Y/T DKO#2 CYR61), p=0.0782 (Y/T DKO#2 CTGF).


J CYR61 CTGF gene expression induced by hydrostatic pressure in ( I ) of Y / T DKO # 1 # 2 normalised to WT . Kruskal - Wallis with Dunn ' s post hoc . * p = 0 . 0259 ( Y / T DKO # 1 CYR61 ) , * * p = 0 . 0007 ( Y / T DKO # 1 CTGF ) , * p = 0 . 0155 ( Y / T DKO # 2 CYR61 ) , * * * p < 0 . 001 ( Y / T DKO # 2 CTGF ) .

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J CYR61 and CTGF gene expression levels induced by hydrostatic pressure in (I) of Y/T DKO #1 and #2 normalised to WT. Kruskal-Wallis test with Dunn's post hoc. *p=0.0259 (Y/T DKO#1 CYR61), **p=0.0007 (Y/T DKO#1 CTGF), *p=0.0155 (Y/T DKO#2 CYR61), ***p<0.001 (Y/T DKO#2 CTGF).


A volume of WT Y / T DKO clones at steady state measured using DHM . Each dot represents a single . Error bars represent mean ± 95 % CI . Kruskal - Wallis with Dunn ' s post - hoc . Data from three independent experiments . * * * p < 0 . 001 , p = 0 . 0917 ( Y / T DKO # 1 vs # 2 ) .

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A Cell volume of WT and Y/T DKO clones at steady state measured using DHM. Each dot represents a single cell. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. Data from three independent experiments. ***p<0.001, p=0.0917 (Y/T DKO #1 vs #2).


B Comparison of volume of WT LATS1 / 2 DKO clones at steady state obtained using DHM . Each dot represents a single . Data from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis with Dunn ' s post - hoc . * * p = 0 . 0018 ( WT vs LATS1 / 2 DKO # 1 ) , * * * p < 0 . 001 ( WT vs LATS1 / 2 DKO # 2 ) , p = 0 . 0557 ( LATS1 / 2 DKO # 1 vs # 2 ) .

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B Comparison of cell volume of WT and LATS1/2 DKO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. **p=0.0018 (WT vs LATS1/2 DKO #1), ***p<0.001 (WT vs LATS1/2 DKO #2), p=0.0557 (LATS1/2 DKO #1 vs #2).


C Comparison of volume of WT MST1 / 2 DKO at steady state obtained using DHM . Each dot represents a single . Data from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U . p = 0 . 2731 .

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C Comparison of cell volume of WT and MST1/2 DKO at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. p=0.2731.


D HEK293A NF2 KO response to 100mbar cyclic hydrostatic pressure . Each dot represents a single Error bars represent mean ± 95 % CI . Data pooled from three independent experiments . Mann - Whitney U . p = 0 . 4072 .

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D HEK293A NF2 KO cells response to 100mbar cyclic hydrostatic pressure. Each dot represents a single cell and Error bars represent mean ± 95% CI. Data pooled from three independent experiments. Mann-Whitney U test. p=0.4072.


E Comparison of volume of WT TEAD KO clones at steady state obtained using DHM . Each dot represents a single . Data from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis with Dunn ' s post - hoc . p > 0 . 9999 for comparisons .

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E Comparison of cell volume of WT and TEAD KO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. p>0.9999 for all comparisons.


F Maxima minima are identified to calculate average change in volume in response to hydrostatic pressure using measurements obtained by DHM .

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F Maxima and minima are identified to calculate average percentage change in cell volume in response to hydrostatic pressure using measurements obtained by DHM.


G Representative WT Y / T DKO single volume change in response to hydrostatic pressure recorded by DHM .

G Representative WT and Y/T DKO single cell volume change in response to hydrostatic pressure recorded by DHM.


H Average change in volume in response to hydrostatic pressure in WT Y / T DKO clones . Each dot represents a single . data were acquired at onset of oscillating pressure . Data from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis with Dunn ' s post hoc . * * * p < 0 . 001 ( for both comparisons ) , p = 0 . 1450 ( Y / T DKO # 1 vs # 2 ) .

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H Average change in cell volume in response to hydrostatic pressure in WT and Y/T DKO clones. Each dot represents a single cell. Images and data were acquired at the onset of oscillating pressure. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post hoc. ***p < 0.001 (for both comparisons), p=0.1450 (Y/T DKO #1 vs #2).


I Representative of Flipper - TR labelled HEK293A WT ( top ) , Y / T DKO # 1 ( middle ) Y / T DKO # 2 ( bottom ) are shown obtained using lifetime imaging microscopy ( FLIM ) ( ex . 485 , em . 600 )

I Representative images of Flipper-TR labelled HEK293A WT (top), Y/T DKO #1 (middle) and Y/T DKO #2 (bottom) are shown obtained using fluorescence lifetime imaging microscopy (FLIM) (ex. 485, em. 600)


J Average lifetime of Flipper - TR probe quantified using lifetime imaging microscope ( FLIM ) ( ex . 485 , em . 600 ) . HEK293A wildtype Y / T DKO clones # 1 # 2 were labelled with 2μM Flipper - TR for 15 minutes prior to imaging . average lifetime measurements were obtained by fitting photon histogram with a double exponential longest lifetime was extracted to report plasma tension , Each dot represents lifetime of a single data shown is pooled from three independent experiments ( mean ± 95 % CI ) . Kruskal - Wallis with Dunn ' s post hoc . * * p = 0 . 0027 ( WT vs Y / T DKO # 1 ) , * p = 0 . 0136 ( WT vs Y / T DKO # 2 ) , p > 0 . 9999 ( Y / T DKO # 1 vs # 2 ) .

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J Average fluorescence lifetime of Flipper-TR probe quantified using fluorescence lifetime imaging microscope (FLIM) (ex. 485, em. 600). HEK293A wildtype and Y/T DKO clones #1 and #2 were labelled with 2μM Flipper-TR for 15 minutes prior to imaging. The average lifetime measurements were obtained by fitting the photon histogram with a double exponential and the longest lifetime was extracted to report plasma membrane tension, Each dot represents fluorescence lifetime of a single cell and data shown is pooled from three independent experiments (mean±95% CI). Kruskal-Wallis test with Dunn's post hoc. **p=0.0027 (WT vs Y/T DKO#1), *p=0.0136 (WT vs Y/T DKO #2), p>0.9999 (Y/T DKO#1 vs #2).


K Representative immunofluorescence showing MYC - tagged YAP expression in WT YAP re - expressing S94A mutant YAP re - expressing compared to Y / T DKO vector control . 95 % were positive for expression of YAP or YAP mutant . Scale bar = 20µm .

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K Representative immunofluorescence images showing MYC-tagged YAP expression in WT YAP re-expressing cells and S94A mutant YAP re-expressing cells compared to Y/T DKO vector control. 95% were positive for expression of YAP or YAP mutant. Scale bar = 20µm.


L Western blot showing total YAP , TAZ CYR61 in WT YAP S94A mutant YAP re - expressing compared to WT Y / T DKO vector control .

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L Western blot showing total YAP, TAZ and CYR61 levels in WT YAP and S94A mutant YAP re-expressing cells compared to WT and Y/T DKO vector control.


M Average change in volume in response to hydrostatic pressure in WT YAP S94A mutant YAP re - expressing relative to Y / T DKO . Each dot represents a single . Error bars represent mean ± 95 % CI . Data pooled from three independent experiments . Kruskal - Wallis with Dunn ' s post hoc . p < 0 . 001 ( vector vs . WT YAP ) , p = 0 . 4912 ( vector vs . S94A YAP ) , p = 0 .

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

M Average change in cell volume in response to hydrostatic pressure in WT YAP and S94A mutant YAP re-expressing cells relative to Y/T DKO. Each dot represents a single cell. Error bars represent mean ± 95% CI. Data pooled from three independent experiments. Kruskal-Wallis test with Dunn's post hoc. p<0.001 (vector vs. WT YAP), p=0.4912 (vector vs. S94A YAP), p=0.0327


N Average change in volume in response to 100mbar cyclic hydrostatic pressure in WT TEAD KO clones . Each dot represents a single . Data pooled from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis with Dunn ' s post hoc . * * * p < 0 . 001 ( for both comparisons ) , p > 0 . 9999 ( TEAD KO # 1 vs # 2 ) .

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

N Average change in cell volume in response to 100mbar cyclic hydrostatic pressure in WT and TEAD KO clones. Each dot represents a single cell. Data pooled from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post hoc. ***p < 0.001 (for both comparisons), p>0.9999 (TEAD KO #1 vs #2).


O WT Y / T DKO volume were measured at steady - state average change in volume was quantified using DHM as in previous experiments , these are labelled ( - ) . were then subjected to cyclic hydrostatic pressure for 2 hours their volume was quantified to determine whether prior exposure to hydrostatic pressure ( ' pre - conditioning ' ) would change their volume response , these are labelled ( + ) . Each dot represents a single error bars represent 95 % CI . Graphs include data obtained from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . ns = p > 0 . 9999 . P average change in volume was quantified under same experimental conditions as in " O " . average change in volume in response to cyclic hydrostatic pressure of with no previous exposure to hydrostatic pressure ( labelled as ( - ) ) was quantified using DHM compared to those with prior exposure to hydrostatic pressure ( labelled ( + ) ) . Each dot represents a single error bars represent 95 % CI . Graphs include data obtained from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . * * p = 0 . 006 ( WT ) , p = 0 . 4173 ( Y / T DKO ) , ns = p > 0 . 9999 .

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

O WT and Y/T DKO cell volume were measured at steady-state and the average change in cell volume was quantified using the DHM as in previous experiments, these are labelled (-). Cells were then subjected to cyclic hydrostatic pressure for 2 hours and their cell volume was quantified to determine whether prior exposure to hydrostatic pressure ('pre-conditioning') would change their cell volume response, these are labelled (+). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal-Wallis with Dunn's post hoc. ns= p>0.9999. P The average percentage change in cell volume was quantified under the same experimental conditions as in "O". The average change in cell volume in response to cyclic hydrostatic pressure of cells with no previous exposure to hydrostatic pressure (labelled as (-)) was quantified using DHM and compared to those with prior exposure to hydrostatic pressure (labelled (+)). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal-Wallis with Dunn's post hoc. **p=0.006 (WT), p=0.4173 (Y/T DKO), ns= p>0.9999.


C Representative immunofluorescence showing Phalloidin - labelled actin ( red ) Hoechst ( blue ) in Latrunculin B ( LatB ) ( 0 . 5µM ) or Cytochalasin D ( CytD ) ( 2µM ) - treated HEK293A compared to control across Hippo pathway genome - edited as shown . Scale bar = 20μm . D Average change in volume obtained by DHM in response to hydrostatic pressure with 2µM Cytochalasin D ( CytD ) treatment in WT , Y / T DKO LATS1 / 2 DKO . Each dot represents a single error bars represent mean ± 95 % CI . Data pooled from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . * * p = 0 . 0044 ( WT con vs CytD ) , p * = 0 . 0409 ( Y / T DKO con vs CytD ) , p > 0 . 9999 ( LATS1 / 2 DKO con vs CytD ) , * * * p = 0 . 0003 ( WT con vs Y / T DKO con ) , * * * p < 0 . 001 ( WT con vs Y / T DKO CytD ) , * * * p < 0 . 001 ( Y / T DKO CytD vs LATS1 / 2 DKO CytD ) . E Fold difference in average change in volume in response to dynamic hydrostatic pressure from ( H ) normalised against untreated . Each dot represents a single error bars are 95 % CI . Mann - Whitney U . * * p = 0 . 0069 ( WT vs . Y / T DKO ) , * * p = 0 . 0031 ( WT vs LATS1 / 2 DKO ) . F Average change in volume in response to hydrostatic pressure with 0 . 5µM Latrunculin B ( Lat B ) treatment in WT , Y / T DKO LATS1 / 2 DKO . Each dot represents a single error bars represent mean ± 95 % CI . Data pooled from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . * * * p < 0 . 001 ( comparisons ) , p = 0 . 0972 ( LATS / 12 DKO con vs LatB ) , * p = 0 . 0216 ( Y / T DKO LatB vs LATS1 / 2 DKO LatB ) . G Fold difference in average change in volume in response to cyclic 0 . 1Hz 100mbar hydrostatic pressure from ( F ) normalised against control . Each dot represents a single error bars represent mean ± 95 % CI . Mann - Whitney U . p = 0 . 1472 ( WT vs . Y / T DKO ) , * * p = 0 . 0015 ( WT vs LATS1 / 2 DKO ) .

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

C Representative immunofluorescence images showing Phalloidin-labelled actin (red) and Hoechst (blue) in Latrunculin B (LatB) (0.5µM) or Cytochalasin D (CytD) (2µM)-treated HEK293A cells compared to control across Hippo pathway genome-edited cells as shown. Scale bar = 20μm. D Average change in cell volume obtained by DHM in response to hydrostatic pressure with 2µM Cytochalasin D (CytD) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. **p=0.0044 (WT con vs CytD), p*=0.0409 (Y/T DKO con vs CytD), p>0.9999 (LATS1/2 DKO con vs CytD), ***p=0.0003 (WT con vs Y/T DKO con), ***p<0.001 (WT con vs Y/T DKO CytD), ***p<0.001 (Y/T DKO CytD vs LATS1/2 DKO CytD). E Fold difference in average change in cell volume in response to dynamic hydrostatic pressure from (H) normalised against untreated cells. Each dot represents a single cell and error bars are 95% CI. Mann-Whitney U test. **p=0.0069 (WT vs. Y/T DKO), **p=0.0031 (WT vs LATS1/2 DKO). F Average change in cell volume in response to hydrostatic pressure with 0.5µM Latrunculin B (Lat B) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. ***p<0.001 (all comparisons), p=0.0972 (LATS/12 DKO con vs LatB), *p=0.0216 (Y/T DKO LatB vs LATS1/2 DKO LatB). G Fold difference in average change in cell volume in response to cyclic 0.1Hz 100mbar hydrostatic pressure from (F) normalised against control. Each dot represents a single cell and error bars represent mean ± 95% CI. Mann-Whitney U test. p=0.1472 (WT vs. Y/T DKO), **p=0.0015 (WT vs LATS1/2 DKO).


A Confocal IF labelled with Hoechst ( Blue ) internalised Transferrin ( Trf ) . were allowed to internalize for ten minutes , fixed acid stripped to remove surface bound Trf . Hydrostatic pressure ( H . P . ) promotes transferrin uptake in WT LATS1 / 2 DKO . Scale bar = 20µm . B of transferrin uptake in WT , Y / T DKO LATS1 / 2 DKO treated with hydrostatic pressure compared to steady state . Each dot represents a single error bars represent mean ± 95 % CI . Data from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . Steady - state statistical is shown in blue , transferrin uptake induced by pressure is shown in black . * * * p < 0 . 001 ( WT con vs + H . P . ) , p = 0 . 2375 ( Y / T DKO con vs + H . P . ) , * * * p < 0 . 001 ( LATS1 / 2 DKO con vs + H . P . ) , p = 0 . 2764 ( WT con vs Y / T DKO con ) , p = 0 . 0808 ( WT con vs LATS1 / 2 DKO con ) , * * * p = 0 . 0004 ( Y / T DKO con vs LATS1 / 2 DKO con ) .

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

A Confocal IF images labelled with Hoechst (Blue) and internalised Transferrin (Trf). Cells were allowed to internalize for ten minutes, fixed and acid stripped to remove surface bound Trf. Hydrostatic pressure (H.P.) promotes transferrin uptake in WT and LATS1/2 DKO cells. Scale bar = 20µm. B Quantification of transferrin uptake in WT, Y/T DKO and LATS1/2 DKO cells treated with hydrostatic pressure compared to steady state. Each dot represents a single cell and error bars represent mean ± 95% CI. Data from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. Steady-state statistical analysis is shown in blue, transferrin uptake induced by pressure is shown in black. ***p<0.001 (WT con vs +H.P.), p=0.2375 (Y/T DKO con vs +H.P.), ***p<0.001 (LATS1/2 DKO con vs +H.P.), p=0.2764 (WT con vs Y/T DKO con), p=0.0808 (WT con vs LATS1/2 DKO con), ***p=0.0004 (Y/T DKO con vs LATS1/2 DKO con).


C Confocal IF myc tagged AP180C ( green ) showing internalised Trf ( red ) stained with Hoechst ( blue ) . AP180C inhibits transferrin uptake in WT Y / T DKO . Scale bar = 20µm . D of transferrin uptake in AP180C positive compared to vector control from as in C . Each dot represents a single from four independent experiments error bars represent mean ± 95 % CI . Mann - Whitney U . * * * p < 0 . 001 ( for both comparisons ) .

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

C Confocal IF images myc tagged AP180C (green) cells showing internalised Trf (red) and stained with Hoechst (blue). AP180C inhibits transferrin uptake in WT and Y/T DKO cells. Scale bar = 20µm. D Quantification of transferrin uptake in AP180C positive cells compared to vector control from images as in C. Each dot represents a single cell from four independent experiments and error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001 (for both comparisons).


E Confocal IF of HEK293A WT showing subcellular localisation of YAP ( red ) in AP180C ( green ) negative positive WT LATS1 / 2 DKO . also stained with Hoechst ( blue ) . F of - to - of YAP in AP180C positive WT LATS1 / 2 DKO compared to vector control from as in E . Each dot represents a single pooled from four independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U . * * * p = 0 . 0008 ( WT ) , p = 0 . 9318 ( LATS1 / 2 DKO ) .

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

E Confocal IF images of HEK293A WT cells showing subcellular localisation of YAP (red) in AP180C (green) negative and positive WT and LATS1/2 DKO cells. Cells also stained with Hoechst (blue). F Quantification of cytoplasmic-to-nuclear ratio of YAP in AP180C positive WT and LATS1/2 DKO cells compared to vector control from images as in E. Each dot represents a single cell pooled from four independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p=0.0008 (WT), p=0.9318 (LATS1/2 DKO).


A , B X - gal showed NB - 3 / LacZ expression in sham - operated ( A ) injured ( B ) spinal cords from adult NB - 3 + / − mice .

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

A, B X-gal staining showed NB-3/LacZ expression in the sham-operated (A) and injured (B) spinal cords from adult NB-3+/−mice.


C , D Western blot ( C ) ( D ) of NB - 3 expression in lesion post - injury . NB - 3 expression were normalized by b - tubulin . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - . Data were analyzed from 10 independent experiments each including 2 mice per group .

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

C, D Western blot analysis (C) and quantification (D) of NB-3 expression in the lesion area post-injury. NB-3 expression levels were normalized by b-tubulin. Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. Data were analyzed from 10 independent experiments each including 2 mice per group.


E , F Single - labeling immunostaining showed NB - 3 expression in sham - operated ( E ) injured ( F ) spinal cords from adult NB - 3 + / + mice .

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

E, F Single-labeling immunostaining showed NB-3 expression in the sham-operated (E) and injured (F) spinal cords from adult NB-3+/+mice.


G - K Co - immunostaining of NB - 3 ( green ) , PDGFR - β ( red ) , GFAP ( blue ) on serial sagittal sections from same NB - 3 + / + spinal cord 14 days post - injury . ( G1 - K1 ) High - magnification of boxed areas in ( G - K ) , respectively . L Co - immunostaining of NB - 3 ( green ) , PDGFR - β ( red ) , GFAP ( blue ) on sagittal sections from NB - 3 − / − spinal cord 14 days post - injury . ( L1 ) High - magnification image of boxed in ( L ) . arrows indicate lesion sites .

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

G-K Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+spinal cord 14 days post-injury. (G1-K1) High-magnification images of boxed areas in (G-K), respectively.L Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (L1) High-magnification image of the boxed area in (L). The arrows indicate the lesion sites.


M - Q Co - immunostaining of NB - 3 ( green ) , Fibronectin ( red ) , GFAP ( blue ) on serial sagittal sections from same NB - 3 + / + spinal cord 14 days post - injury . ( M1 - Q1 ) High - magnification of boxed areas in ( M - Q ) , respectively . R Co - immunostaining of NB - 3 ( green ) , Fibronectin ( red ) , GFAP ( blue ) on sagittal sections from NB - 3 − / − spinal cord 14 days post - injury . ( R1 ) High - magnification image of boxed in ( R ) . arrows in ( G - R ) indicate lesion sites .

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

M-Q Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+spinal cord 14 days post-injury. (M1-Q1) High-magnification images of boxed areas in (M-Q), respectively.R Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (R1) High-magnification image of the boxed area in (R). The arrows in (G-R) indicate the lesion sites.


B NB - 3 expression in corticospinal neurons 14 28 days post - injury ( dpi ) . FG ( blue ) was applied to retrogradely label corticospinal neurons . C of of NB - 3 in ( B ) . * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - . intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

B NB-3 expression in corticospinal neurons 14 and 28 days post-injury (dpi). FG (blue) was applied to retrogradely label corticospinal neurons.C Quantification of the fluorescence intensity of NB-3 in (B). **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


D , E Western blot ( D ) ( E ) of NB - 3 expression in sensorimotor cortex after SCI . NB - 3 expression were normalized by b - tubulin . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - . Data were analyzed from 3 independent experiments each including 3 mice per group .

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

D, E Western blot analysis (D) and quantification (E) of NB-3 expression in the sensorimotor cortex after SCI. NB-3 expression levels were normalized by b-tubulin. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.


F - K Induced NB - 3 expression at distal terminations of post - traumatic corticospinal axons in reactive astrocytes at lesion border 14 ( H I ) 28 days post - injury ( dpi ) ( J K ) . BDA ( red ) was applied to anterogradely label corticospinal axons . GFAP ( blue ) was used to label reactive astrocytes . ( G ) High - magnification image of boxed in ( F ) . ( I K ) High - magnification x - y - z reconstruction of boxed areas in ( H J ) , respectively , show co - localization of NB - 3 GFAP in astrocytes at lesion border co - localization of NB - 3 in BDA - labeled axons GFAP - positive astrocytes at contact surface of corticospinal axons astrocytes ( arrowheads ) . arrows in ( H J ) indicate lesion sites . asterisks in ( H J ) indicate terminations of BDA - labeled projecting axons .

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

F-K Induced NB-3 expression at the distal terminations of post-traumatic corticospinal axons and in reactive astrocytes at the lesion border 14 (H and I) and 28 days post-injury (dpi) (J and K). BDA (red) was applied to anterogradely label corticospinal axons. GFAP (blue) was used to label reactive astrocytes. (G) High-magnification image of the boxed area in (F). (I and K) High-magnification and x-y-z reconstruction images of boxed areas in (H and J), respectively, show co-localization of NB-3 and GFAP in astrocytes at the lesion border and co-localization of NB-3 in BDA-labeled axons and GFAP-positive astrocytes at the contact surface of corticospinal axons and astrocytes (arrowheads). The arrows in (H and J) indicate the lesion sites. The asterisks in (H and J) indicate the terminations of the BDA-labeled projecting axons.


B - K Representative of serial sagittal sections showing main BDA - labeled corticospinal axons in NB - 3 + / + ( B - D ) NB - 3 − / − ( E - G ) mice 14 after complete spinal transection . ( H - J ) High - magnification of areas in ( E - G ) ( single asterisks ) , respectively . ( K ) High - magnification image of in ( E ) ( double asterisk ) . arrows indicate lesion sites ; dashed lines indicate regenerative corticospinal axons extending into distal spinal cord ; single asterisks in ( E - G ) indicate lesion epicenters ; double asterisk in ( E ) indicates axons extending from caudal lesion border . Scale bars , 400 µm ( B - G ) 100 µm ( H - K ) . L of index of BDA - labeled axons at certain distances from lesion border in ( B - G ) . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 25 mice per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K Representative images of serial sagittal sections showing the main BDA-labeled corticospinal axons in NB-3+/+ (B-D) and NB-3−/− (E-G) mice 14 weeks after complete spinal transection. (H-J) High-magnification images of the areas in (E-G) (single asterisks), respectively. (K) High-magnification image of the area in (E) (double asterisk). The arrows indicate the lesion sites; the white dashed lines indicate the regenerative corticospinal axons extending into the distal spinal cord; the single asterisks in (E-G) indicate the lesion epicenters; the double asterisk in (E) indicates the axons extending from the caudal lesion border. Scale bars, 400 µm (B-G) and 100 µm (H-K).L Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (B-G). Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 25 mice per group.


A Time course of change in BMS scores in NB - 3 + / + NB - 3 − / − mice as measured with BMS rating scale after spinal cord complete transection . * p < 0 . 05 ; two - way ANOVA followed by Bonferroni ' s post - . n = 25 mice per group .

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

A Time course of the change in BMS scores in NB-3+/+ and NB-3−/−mice as measured with the BMS rating scale after spinal cord complete transection. *p < 0.05; two-way ANOVA followed by Bonferroni's post-test. n= 25 mice per group.


C Electrophysiology transmission across spinal lesion site . In each group of NB - 3 + / + NB - 3 − / − mice , electrophysiological responses in caudal spinal cord were recorded in condition of sham - operated or 12 post - injury . latencies amplitudes of responses were analyzed quantified in each group . n . s . , not significant , * p < 0 . 05 , * * p < 0 . 01 ; two - way ANOVA followed by Bonferroni ' s post - . n = 10 mice per group .

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

C Electrophysiology transmission across the spinal lesion site. In each group of NB-3+/+ and NB-3−/−mice, the electrophysiological responses in the caudal spinal cord were recorded in the condition of sham-operated or 12 weeks post-injury. The latencies and amplitudes of responses were analyzed and quantified in each group. n.s., not significant, *p < 0.05, and **p < 0.01; two-way ANOVA followed by Bonferroni's post-test. n= 10 mice per group.


E Representative of coronal sections showing synapse formation between glutamatergic corticospinal terminals spinal neurons in NB - 3 + / + NB - 3 − / − mice . Corticospinal axons , neurons , glutamatergic terminals of corticospinal axons were labeled with BDA ( red ) , Tuj1 ( blue ) vGlut1 / 2 ( green ) , respectively . Each group showed data from rostral caudal spinal cord in condition of sham - operated or 12 post - injury . Scale bar , 20 μm . F of number of glutamatergic corticospinal synapses per neuron in each group . n . s . , not significant , * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 15 mice per group .

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

E Representative images of coronal sections showing synapse formation between the glutamatergic corticospinal terminals and spinal neurons in NB-3+/+ and NB-3−/−mice. Corticospinal axons, neurons, and glutamatergic terminals of corticospinal axons were labeled with BDA (red), Tuj1 (blue) and vGlut1/2 (green), respectively. Each group showed the data from the rostral and caudal spinal cord in the condition of sham-operated or 12 weeks post-injury. Scale bar, 20 μm.F Quantification of the number of glutamatergic corticospinal synapses per neuron in each group. n.s., not significant, **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 15 mice per group.


A , B Western blot ( A ) ( B ) of NB - 3 expression in cultured pyramidal neurons infected with lentivirus encoding negative control ( LV - nc - GFP ) that encoding NB - 3 shRNA ( LV - NB - 3 shRNA - GFP ) . NB - 3 expression were normalized by b - tubulin . * * * p < 0 . 001 ; one sample t - . Data were analyzed from 5 independent experiments .

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

A, B Western blot analysis (A) and quantification (B) of NB-3 expression in cultured pyramidal neurons infected with lentivirus encoding negative control (LV-nc-GFP) and that encoding NB-3 shRNA (LV-NB-3 shRNA-GFP). NB-3 expression levels were normalized by b-tubulin. ***p < 0.001; one sample t-test. Data were analyzed from 5 independent experiments.


C - E Sagittal sections showing BDA - labeled corticospinal axons in injured spinal cords following cortical injection of LV - nc - GFP ( C ) LV - NB - 3 shRNA - GFP ( D ) . High - magnification image ( E ) of in ( D ) ( asterisk ) . F , J of index of BDA - labeled axons at certain distances from lesion border in ( C , D , G , H ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 19 mice per group in ( F ) , n = 17 mice per group in ( J ) .

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C-E Sagittal sections showing the BDA-labeled corticospinal axons in injured spinal cords following cortical injection of LV-nc-GFP (C) and LV-NB-3 shRNA-GFP (D). High-magnification image (E) of the area in (D) (asterisk).F, J Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (C, D, G, and H). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 19 mice per group in (F), and n= 17 mice per group in (J).


G - I Sagittal sections showing corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( G ) LV - NB - 3 shRNA - GFP ( H ) into lesion sites . High - magnification image ( I ) of in ( H ) ( asterisk ) . arrows in ( C - E G - H ) indicate lesion sites ; asterisks in ( D H ) indicate lesion epicenters ; dashed lines in ( D H ) indicate regenerative corticospinal axons extending into distal spinal cord . F , J of index of BDA - labeled axons at certain distances from lesion border in ( C , D , G , H ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 19 mice per group in ( F ) , n = 17 mice per group in ( J ) .

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

G-I Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (G) and LV-NB-3 shRNA-GFP (H) into the lesion sites. High-magnification image (I) of the area in (H) (asterisk). The arrows in (C-E and G-H) indicate the lesion sites; the asterisks in (D and H) indicate the lesion epicenters; the white dashed lines in (D and H) indicate the regenerative corticospinal axons extending into the distal spinal cord.F, J Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (C, D, G, and H). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 19 mice per group in (F), and n= 17 mice per group in (J).


A , B NB - 3 - Fc - coated bead aggregation ( A ) . BSA - coated beads as a negative control ( B ) .

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A, B NB-3-Fc-coated bead aggregation assay (A). BSA-coated beads as a negative control (B).


C Co - immunoprecipitation of NB - 3 - Myc with NB - 3 - HA from co - transfected COS1 .

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

C Co-immunoprecipitation of NB-3-Myc with NB-3-HA from co-transfected COS1 cells.


D , E Cis interactions of NB - 3 - Myc with NB - 3 - HA in COS1 . ( E ) High - magnification image of boxed in ( D ) . were co - stained for Myc ( red ) , HA ( green ) , DAPI ( blue ) . arrowheads indicate surface co - localization of NB - 3 - Myc NB - 3 - HA .

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

D, E Cis interactions of NB-3-Myc with NB-3-HA in COS1 cells. (E) High-magnification image of the boxed area in (D). Cells were co-stained for Myc (red), HA (green), and DAPI (blue). The arrowheads indicate cell surface co-localization of NB-3-Myc and NB-3-HA.


F , G Trans interactions between NB - 3 expressed in COS1 treated NB - 3 - Fc . were co - stained for Myc ( red ) , Fc or NB - 3 - Fc ( green ) , DAPI ( blue ) . arrows indicate co - localization of NB - 3 - Myc NB - 3 - Fc at ( G ) .

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

F, G Trans interactions between NB-3 expressed in COS1 cells and treated NB-3-Fc. Cells were co-stained for Myc (red), Fc or NB-3-Fc (green), and DAPI (blue). The arrows indicate co-localization of NB-3-Myc and NB-3-Fc at the cell membrane (G).


H co - localization of NB - 3 - Myc NB - 3 - HA separately expressed in 293T COS1 . were co - stained for Myc ( red ) , HA ( green ) , DAPI ( blue ) .

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

H Cell membrane co-localization of NB-3-Myc and NB-3-HA separately expressed in 293T and COS1 cells. Cells were co-stained for Myc (red), HA (green), and DAPI (blue).


I , J NB - 3 expression in cultured pyramidal neurons astrocytes . Pyramidal neurons were co - stained for NB - 3 , Ctip2 Tuj1 ( I ) . Astrocytes were co - stained for NB - 3 , GFAP , DAPI ( J ) .

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

I, J NB-3 expression in cultured pyramidal neurons and astrocytes. Pyramidal neurons were co-stained for NB-3, Ctip2 and Tuj1 (I). Astrocytes were co-stained for NB-3, GFAP, and DAPI (J).


K Western blot of NB - 3 in cultured pyramidal neurons astrocytes . NB - 3 expression were normalized by b - tubulin expressed relative to that of neurons . * * * p < 0 . 001 ; one sample t - . Data were analyzed from 3 independent experiments .

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

K Western blot analysis and quantification of NB-3 in cultured pyramidal neurons and astrocytes. NB-3 expression levels were normalized by b-tubulin and expressed relative to that of neurons. ***p < 0.001; one sample t-test. Data were analyzed from 3 independent experiments.


L - Q NB - 3 + / + ( L M ) NB - 3 − / − ( O P ) pyramidal neurons were co - cultured with astrocytes that did not ( L O ) or did ( M P ) express NB - 3 , immunostained for Tuj1 , NB - 3 , GFAP ( L , M , O P ) . NB - 3 + / + pyramidal neurons ( N ) NB - 3 − / − pyramidal neurons that overexpressed NB - 3ΔECD - Myc ( Q ) were co - cultured with astrocytes that overexpressed NB - 3ΔECD - Myc , immunostained for Tuj1 , Myc , GFAP ( N Q ) . R of neurites on astrocytes relative to total neurite . n . s . , not significant , * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - . neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified .

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

L-Q NB-3+/+ (L and M) and NB-3−/− (O and P) pyramidal neurons were co-cultured with astrocytes that did not (L and O) or did (M and P) express NB-3, and immunostained for Tuj1, NB-3, and GFAP (L, M, O and P). NB-3+/+ pyramidal neurons (N) and NB-3−/−pyramidal neurons that overexpressed NB-3ΔECD-Myc (Q) were co-cultured with astrocytes that overexpressed NB-3ΔECD-Myc, and immunostained for Tuj1, Myc, and GFAP (N and Q).R The length of neurites on astrocytes relative to total neurite length. n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified.


A Layer V cortex explants from E18 . 5 NB - 3 + / + NB - 3 − / − mice were cultured in medium supplemented with either Fc or NB - 3 - Fc ( 50 μg / ml ) . B of neurite outgrowth in ( A ) . n . s . , not significant , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - . Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group .

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A Layer V cortex explants from E18.5 NB-3+/+ and NB-3−/−mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml).B Quantification of neurite outgrowth in (A). n.s., not significant, **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group.


C Pyramidal neurons of NB - 3 + / + NB - 3 − / − mice were cultured in medium supplemented with either Fc or NB - 3 - Fc ( 50 μg / ml ) . neurons were co - stained for Ctip2 , NB - 3 , Rhodamine phalloidin . ( C1 - C4 ) High - magnification of growth cones ( arrowheads ) of neurons in ( C ) , respectively . D of neurite in ( C ) , of collapsed growth cones in ( C ) . n . s . , not significant , * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - . Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group .

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

C Pyramidal neurons of NB-3+/+ and NB-3−/− mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml). The neurons were co-stained for Ctip2, NB-3, and Rhodamine phalloidin. (C1-C4) High-magnification images of the growth cones (white arrowheads) of the neurons in (C), respectively.D Quantification of neurite length in (C), and the percentage of collapsed growth cones in (C). n.s., not significant, *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group.


A of coronal sections of layer Vsensorimotor cortex from NB - 3 + / + NB - 3 − / − mice after SCI . Corticospinal neurons were retrogradely labeled with Fluorogold ( FG ) , coronal sections were co - stained with p - mTOR ( A ) ( arrowheads ) . B of of p - mTOR in FG - labeled corticospinal neurons in ( A ) . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - . intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

A Images of coronal sections of layer Vsensorimotor cortex from NB-3+/+ and NB-3−/−mice after SCI. Corticospinal neurons were retrogradely labeled with Fluorogold (FG), and coronal sections were co-stained with p-mTOR (A) (arrowheads).B Quantification of fluorescence intensity of p-mTOR in FG-labeled corticospinal neurons in (A). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


C of coronal sections of layer Vsensorimotor cortex from NB - 3 + / + NB - 3 − / − mice after SCI . Corticospinal neurons were retrogradely labeled with Fluorogold ( FG ) , coronal sections were co - stained with p - S6 ( S240 / 244 ) ( C ) ( arrowheads ) . D of of p - S6 ( S240 / 244 ) in FG - labeled corticospinal neurons in ( C ) . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - . intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

C Images of coronal sections of layer Vsensorimotor cortex from NB-3+/+ and NB-3−/−mice after SCI. Corticospinal neurons were retrogradely labeled with Fluorogold (FG), and coronal sections were co-stained with p-S6 (S240/244) (C) (arrowheads).D Quantification of fluorescence intensity of p-S6 (S240/244) in FG-labeled corticospinal neurons in (C). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


E - H Western blot of expression of p - Akt / Akt ( E F ) p - S6 ( S240 / 244 ) ( G H ) in sensorimotor cortex from NB - 3 + / + NB - 3 − / − mice after SCI . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - . Data were analyzed from 3 independent experiments each including 3 mice per group .

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E-H Western blot analysis and quantification of the expression of p-Akt/Akt (E and F) and p-S6 (S240/244) (G and H) in sensorimotor cortex from NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.


I , K , M , O Expression of Akt ( I ) , p - Akt ( K ) , p - mTOR ( M ) , p - S6 ( S240 / 244 ) ( O ) in NB - 3 + / + pyramidal neuron - astrocyte co - cultures . astrocytes were transfected either with NB - 3 - Myc or a control vector . dashed lines outline astrocytes . asterisks indicate NB - 3 - expressing astrocytes . J , L , N , P of of Akt ( J ) , p - Akt ( L ) , p - mTOR ( N ) , p - S6 ( S240 / 244 ) ( P ) , respectively , in pyramidal neurons of co - cultures in ( I , K , M , O ) . n . s . , not significant , * p < 0 . 05 ; one sample t - . intensities of more than 300 pyramidal neurons from 6 independent experiments in each group were quantified .

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

I, K, M, O Expression of Akt (I), p-Akt (K), p-mTOR (M), and p-S6 (S240/244) (O) in NB-3+/+ pyramidal neuron-astrocyte co-cultures. The astrocytes were transfected either with NB-3-Myc or a control vector. The dashed lines outline the astrocytes. The asterisks indicate the NB-3-expressing astrocytes.J, L, N, P Quantification of fluorescence intensity of Akt (J), p-Akt (L), p-mTOR (N), and p-S6 (S240/244) (P), respectively, in pyramidal neurons of the co-cultures in (I, K, M, and O). n.s., not significant, and *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 6 independent experiments in each group were quantified.


A Co - immunostaining of CHL1 ( blue ) , NB - 3 ( green ) , BDA ( red ) in injured spinal cords of NB - 3 + / + ( A1 ) NB - 3 − / − ( A5 ) mice . ( A2 - A4 A6 - A8 ) High - magnification of boxed areas in ( A1 A5 ) , respectively . in ( A2 A6 ) are presented as single - channel in ( A3 - A4 A7 - A8 ) , respectively . B of of CHL1 at spinal lesion sites of NB - 3 + / + NB - 3 − / − mice after SCI . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - . n = 11 mice per group .

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

A Co-immunostaining of CHL1 (blue), NB-3 (green), and BDA (red) in injured spinal cords of NB-3+/+ (A1) and NB-3−/− (A5) mice. (A2-A4 and A6-A8) High-magnification images of boxed areas in (A1 and A5), respectively. Images in (A2 and A6) are presented as single-channel fluorescence images in (A3-A4 and A7-A8), respectively.B Quantification of fluorescence intensity of CHL1 at the spinal lesion sites of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. n = 11 mice per group.


C , D Western blot ( C ) ( D ) of CHL1 expression in spinal lesion areas of NB - 3 + / + NB - 3 − / − mice after SCI . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA followed by Bonferroni post - . Data were analyzed from 3 independent experiments each including 2 mice per group .

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

C, D Western blot analysis (C) and quantification (D) of CHL1 expression in spinal lesion areas of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 2 mice per group.


E Co - immunoprecipitation of NB - 3 CHL1 from spinal cord lesion areas of NB - 3 + / + NB - 3 − / − mice .

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

E Co-immunoprecipitation of NB-3 and CHL1 from spinal cord lesion areas of NB-3+/+ and NB-3−/−mice.


F Co - immunostaining of CHL1 NB - 3 in FG - labeled corticospinal neurons ( arrowheads ) of NB - 3 + / + NB - 3 − / − mice 14 days post - injury ( dpi ) . G of of CHL1 in ( F ) . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - . intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

F Co-immunostaining of CHL1 and NB-3 in FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi).G Quantification of the fluorescence intensity of CHL1 in (F). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


H , I Western blot ( H ) ( I ) of CHL1 expression in sensorimotor cortices of NB - 3 + / + NB - 3 − / − mice after SCI . * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - . Data were analyzed from 3 independent experiments each including 3 mice per group .

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

H, I Western blot analysis (H) and quantification (I) of CHL1 expression in sensorimotor cortices of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.


J CHL1 expression in somas axons of NB - 3 + / + NB - 3 − / − pyramidal neurons when co - culturing with NB - 3 - expressing astrocytes ( dashed lines asterisks ) or astrocytes that didn ' t express NB - 3 ( dashed lines ) . Co - localization of CHL1 NB - 3 in astrocytes overexpressed NB - 3 ( asterisks ) . Co - localization of CHL1 NB - 3 in somas axons of co - cultured NB - 3 + / + pyramidal neurons ( arrowheads ) . Left inset , high - magnification view of soma ( arrowhead ) . Right inset , high - magnification view of axon tip ( green arrowhead ) . K of of CHL1 in ( J ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA followed by Bonferroni post - . intensities of more than 300 astrocytes or pyramidal neurons from 3 independent experiments in each group were quantified .

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

J CHL1 expression in the somas and axons of NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3-expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Co-localization of CHL1 and NB-3 in astrocytes overexpressed NB-3 (asterisks). Co-localization of CHL1 and NB-3 in the somas and axons of co-cultured NB-3+/+ pyramidal neurons (arrowheads). Left inset, high-magnification view of the soma (white arrowhead). Right inset, high-magnification view of the axon tip (green arrowhead).K Quantification of fluorescence intensity of CHL1 in (J). *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 astrocytes or pyramidal neurons from 3 independent experiments in each group were quantified.