( C ) Larval NSCs of control ( ctrl ; yw ) , pr - set7716 / Df , pr - set7716 / Df with UAS - pr - set7 induced with insc - Gal4 , and pr - set7716 / Df with pr - set7 - BAC at 24h ALH were stained for H4K20me1 , Dpn , Mira and DAPI . Control : n = 108 ; pr - set7716 / Df : n = 87 ; pr - set7716 / Df with UAS - pr - set7 induced with insc - Gal4 : n = 167 and pr - set7716 / Df with pr - set7 - BAC : n = 104 . Data information : White arrows point to NSCs . Scale bars , 5

O O O B-ORGANISM B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O O

(C) Larval NSCs of control (ctrl; yw), pr-set7716/Df, pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4, and pr-set7716/Df with pr-set7-BAC at 24h ALH were stained for H4K20me1, Dpn, Mira and DAPI. Control: n=108; pr-set7716/Df: n=87; pr-set7716/Df with UAS-pr-set7 induced with insc-Gal4: n=167 and pr-set7716/Df with pr-set7-BAC: n=104. Data information: White arrows point to NSCs. Scale bars, 5 μm


( E ) Larval NSCs with FUCCI constructs induced with insc - Gal4 at 24h were stained for GFP , RFP , Dpn , and Prospero ( Pros ) . ( F ) Quantifications of quiescent NSCs in different cell - cycle stages from FUCCI flies ( induced with insc - Gal4 ) in control ( and insc - Gal4 > FUCCI ) and pr - set7716 / Df . Quiescent NSCs of insc - Gal4 > FUCCI : 76 . 1 % ± 1 . 5 % in G2 , 1 . 7 % ± 0 . 2 % in S and 22 . 2 % ± 1 . 3 % in G0 ( n = 343 ) . Quiescent NSCs of insc - Gal4 > FUCCI ; pr - set7716 / Df : 74 . 4 % ± 1 . 0 % in G2 , 0 . 9 % ± 0 . 2 % in S and 24 . 7 % ± 0 . 8 % in G0 ( n = 344 ) . Data information : Quantification data are presented in as mean ± SD Statistical analyses were done comparing two different genotypes using Mann - Whitney test ns : non - significant . White arrows point to NSCs . Scale bars , 5

O O O B-ORGANISM B-CELL_TYPE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O

(E) Larval NSCs with FUCCI constructs induced with insc-Gal4 at 24h were stained for GFP, RFP, Dpn, and Prospero (Pros). (F) Quantifications of quiescent NSCs in different cell-cycle stages from FUCCI flies (induced with insc-Gal4) in control (and insc-Gal4>FUCCI) and pr-set7716/Df. Quiescent NSCs of insc-Gal4>FUCCI: 76.1%±1.5% in G2, 1.7%±0.2% in S and 22.2%±1.3% in G0 (n=343). Quiescent NSCs of insc-Gal4>FUCCI; pr-set7716/Df: 74.4%±1.0% in G2, 0.9%±0.2% in S and 24.7%±0.8% in G0 (n=344). Data information: Quantification data are presented in as mean ± SD Statistical analyses were done comparing two different genotypes using Mann-Whitney test ns: non-significant. White arrows point to NSCs. Scale bars, 5 μm


( D ) qPCR analysis of pr - set7 , cdk1 , ebd1 and arm in larval brains of control ( ctrl ; yw ) and pr - set7716 / Df ( 3R ) ED5644 ( 716 / Df ) at 24h ALH . n = 7 as biological replicates . Statistical analyses were done comparing between control ( GAPDH1 , GAPDH2 , Tbp1 and Rpl32 ) and pr - set7716 / Df , using Mann - Whitney test . The mRNA levels of control were normalized to 1 . Data are presented as mean ± SD calculated from all replicates . Data information : Data are presented in ( D , as mean ± SD . Statistical analyses were done comparing two different genotypes using Mann - Whitney test ( D * P < 0 .

O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of pr-set7, cdk1, ebd1 and arm in larval brains of control (ctrl; yw) and pr-set7716/Df(3R)ED5644 (716/Df) at 24h ALH. n=7 as biological replicates. Statistical analyses were done comparing between control (GAPDH1, GAPDH2, Tbp1 and Rpl32) and pr-set7716/Df, using Mann-Whitney test. The mRNA levels of control were normalized to 1. Data are presented as mean ± SD calculated from all replicates. Data information: Data are presented in (D, as mean ± SD. Statistical analyses were done comparing two different genotypes using Mann-Whitney test (D *P < 0.05


G ) Larval NSCs of control ( ctrl ; yw ) and pr - set7716 / Df at 24h ALH were stained for Ebd1 , Dpn and Mira . Yellow dotted boxes , zoomed - in area . White arrows point to NSCs . Data information : Scale bars , 15 μm ( G ) .

O O B-ORGANISM B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O

G) Larval NSCs of control (ctrl; yw) and pr-set7716/Df at 24h ALH were stained for Ebd1, Dpn and Mira. Yellow dotted boxes, zoomed-in area. White arrows point to NSCs. Data information: Scale bars, 15 μm (G).


( H , I ) Quantifications of nuclear Ebd1 ( H ) and the corrected total cell fluorescence ( CCF ) ( I ) In ( H ) , n = 756 , 9 BL for control ; n = 765 , 9 BL for pr - set7716 / Df . In ( I ) , CCF = Integrated Density - ( Area of selected cell × Mean fluorescence of background readings ) . CCFs were 1102 . 2 ± 493 . 2 ( n = 99 ) for control ( ctrl ; yw ) and 144 . 0 ± 88 . 0 ( n = 98 ) for 716 / Df . Data information : Data are presented in , I , s mean ± SD . n numbers in ( H ) 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 , H , I , * * * P < 0 . 001 .

O O O O O O O B-TISSUE B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O O O O O 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) Quantifications of nuclear Ebd1 (H) and the corrected total cell fluorescence (CCF) (I) In (H), n=756, 9 BL for control; n=765, 9 BL for pr-set7716/Df. In (I), CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 1102.2±493.2 (n=99) for control (ctrl; yw) and 144.0±88.0 (n=98) for 716/Df. Data information: Data are presented in , I, s mean ± SD. n numbers in (H) 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 , H, I, ***P < 0.001.


( 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 -

O O O B-ORGANISM B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O 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-CELL_TYPE O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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 ) 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 .

O O O B-ORGANISM B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O 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-GENEPROD O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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-CELL_TYPE O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O B-ORGANISM B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O 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-CELL_TYPE O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ,

O O O B-ORGANISM B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O 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-CELL_TYPE O O O B-TISSUE I-TISSUE O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O B-EXP_ASSAY O O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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 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-ORGANISM B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O 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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SMALL_MOLECULE 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 and stably expressing full - length ( FL ) EB1 , but not in control Flag or any of the other EB1 constructs depicted in A .

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(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 cells .

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(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 analysis , respectively .

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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 and 0 . 02 ) ( F ) infection is reduced in differentiated THP - 1 depleted of CLIP170 determined by luciferase assays or FACS analysis , respectively .

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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 cells .

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


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 .

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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 cells .

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


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

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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 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 .

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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 ) 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 ) .

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(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 ) 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 .

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(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 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 .

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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 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 .

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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 ) 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 .

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(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 and analyzed by WB Molecular weight markers ( in kDa ) are shown to the right of WBs in A .

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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 intensity of fused viral particles normalized to the control mean .

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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 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 ) .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis demonstrating that while GAPDH - HA control do not bind ( A FEZ1 - Flag control pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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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 analysis demonstrating that while EB1 - Flag do not bind pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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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 analysis demonstrating that while Flag - CLIP170 pelleted with in vitro assembled HIV - 1 CA - NC complexes .

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


( 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 .

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(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 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 .

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(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 ( Virus # 8 and # 9 ) but not with Mock fractions .

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(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 cells stably expressing Flag - CLIP170 , but not control Flag or Flag - CLIP170ΔCAPgly . Molecular weight markers ( in kDa ) are shown to the right of WBs .

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(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 images ( n = 3 ) of CHME3 expressing Flag - CLIP170 or Flag - CLIP170ΔCAPgly stained for Flag and 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

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

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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 cells were subjected to CA - NC binding assays and WB analysis .

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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 ) 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 .

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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 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 .

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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 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 .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O

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 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 .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O

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 ( 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 )

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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 ( 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 .

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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 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 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 .

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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 ) cells expressing GAPDH alongside GAPDH -

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


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 .

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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 cells infected with SIV - VSV - luc ( F ) stained for Ac - MTs , Tyr - MTs , and the 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 cells infected with MuLV - VSV - luc ( G ) stained for Ac - MTs , Tyr - MTs , and the 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 cells infected with SIV - VSV - luc analyzed by

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


Rat2 cells 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 ) cells depleted of CLIP170 . Statistical significance was determined by t test . * * * = p < 0 . 001 . Data are mean values from two independent experiments + / - SEM .

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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 ) cells depleted of CLIP170 .

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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 analysis is shown ( n = 2 ) . Molecular weight markers ( in kDa ) are shown to the 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 - 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 ( - ) .

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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 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 .

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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 - based Western blot probed for YAP reveals increased levels of unphosphorylated ( downshift ) YAP in 143B cells 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 protein levels in control ( - ) and with hydrostatic pressure ( + ) stimulation in 143B cells from cell 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 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 .

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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 and TAZ expression in two independent ( # 1 and # 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 and minima are identified to calculate average percentage change in cell 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 and Y / T DKO single cell volume change in response to hydrostatic pressure recorded by DHM .

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


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 ) .

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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 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 )

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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 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 ) .

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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 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 .

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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 and CYR61 levels in WT YAP and S94A mutant YAP re - expressing cells compared to WT and 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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O B-GENEPROD 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 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 O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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 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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD 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 staining showed NB - 3 / LacZ expression in the sham - operated ( A ) and injured ( B ) spinal cords from adult NB - 3 + / − mice .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM 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 the sham - operated ( E ) and injured ( F ) spinal cords from adult NB - 3 + / + mice .

O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM 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 ) , 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 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O 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-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O 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 ) , 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 .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O 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-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O 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 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 .

O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O O B-ORGANISM 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_TYPE O 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-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O O 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-EXP_ASSAY O O O B-SUBCELLULAR 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 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 .

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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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O 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-ORGANISM 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.