( e ; HA - GluN2Bct - CTMm ; P = 0 . 785 compared to control ( white bar ) ; F ( 4 , 35 ) = 0 . 432 , 8 independent experiments from 8 separate cell cultures and transfections ) . Levels of cDAPK1 or wtDAPK1 cotransfected with pcDNA3 . 0 vector ( 0 : 1 , white bar ) represent the control values , arbitrarily set as 1 . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , compared with the control ; n . s . , not significant . Bars represent relative means ± s . e . m . Full - length blots are presented in Supplementary Figure 9 . Membranes reprobed for β - actin were used as loading controls . One - way ANOVA with Fisher least significant difference test .

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

(e; HA-GluN2Bct-CTMm; P = 0.785 compared to control (white bar); F(4,35) = 0.432, 8 independent experiments from 8 separate cell cultures and transfections). Levels of cDAPK1 or wtDAPK1 cotransfected with pcDNA3.0 vector (0:1, white bar) represent the control values, arbitrarily set as 1. *P 0.05, **P 0.01, ***P 0.001, compared with the control; n.s., not significant. Bars represent relative means ± s.e.m. Full-length blots are presented in Supplementary Figure 9. Membranes reprobed for β-actin were used as loading controls. One-way ANOVA with Fisher least significant difference test.


( a ) Left , NMDA ( 50 μM ; 30 min ) activated DAPK1 , resulting in a time - dependent decrease in its phosphorylation levels ( pDAPK1 , n = 4 ) . Right , coimmunoprecipitation ( IP ) with anti - GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA - induced association between DAPK1 and GluN2B ( n = 3 ) .

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

(a) Left, NMDA (50 μM; 30 min) activated DAPK1, resulting in a time-dependent decrease in its phosphorylation levels (pDAPK1, n = 4). Right, coimmunoprecipitation (IP) with anti-GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA-induced association between DAPK1 and GluN2B (n = 3).


( b ) Design and production of TAT - GluN2Bct - CTM and TAT - GluN2Bct peptides ( left ) using an E . coli expression system . Coomassie blue staining after SDS - PAGE assessed their purity ( right ) ; left lane , size marker .

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

(b) Design and production of TAT-GluN2Bct-CTM and TAT-GluN2Bct peptides (left) using an E. coli expression system. Coomassie blue staining after SDS-PAGE assessed their purity (right); left lane, size marker.


( c ) Bath application of TAT - GluN2Bct - CTM ( 200 μM ; n = 9 ) , but not TAT - GluN2Bct ( 200 μM ; n = 6 ) , knocked down activated DAPK1 , and this was prevented by NH4Cl ( 20 mM ; n = 5 ; one - way ANOVA ; P 0 . 001 , F ( 5 , 36 ) = 10 . 891 ) and dose - ( d , doses in μM ; n = 4 ; P 0 . 001 ; F ( 6 , 21 ) = 18 . 14 ) and time - dependent ( e ; P 0 . 001 ; F ( 8 , 44 ) = 12 . 074 ) . ( f ) A single pretreatment of TAT - GluN2Bct - CTM ( sing ; 200 μM , 60 min before and during the 30 - min NMDA stimulation ) produced a transient reduction of DAPK1 , returning to baseline within 7 h ( n = 4 ; P = 0 . 888 ) , and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h ( mult ; n = 4 ; ΔΔP = 0 . 002 ) . One - way ANOVA ; P 0 . 001 , F ( 4 , 15 ) = 10 . 389 .

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

(c) Bath application of TAT-GluN2Bct-CTM (200 μM; n = 9), but not TAT-GluN2Bct (200 μM; n = 6), knocked down activated DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; one-way ANOVA; P 0.001, F(5,36) = 10.891) and dose- (d, doses in μM; n = 4; P 0.001; F(6,21) = 18.14) and time-dependent (e; P 0.001; F(8,44) = 12.074). (f) A single pretreatment of TAT-GluN2Bct-CTM (sing; 200 μM, 60 min before and during the 30-min NMDA stimulation) produced a transient reduction of DAPK1, returning to baseline within 7 h (n = 4; P = 0.888), and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h (mult; n = 4; ΔΔP = 0.002). One-way ANOVA; P 0.001, F(4,15) = 10.389.


( g ) Synthetic peptides TAT - GluN2B and TAT - GluN2BCTM .

(g) Synthetic peptides TAT-GluN2B and TAT-GluN2BCTM.


( h ) TAT - GluN2BCTM ( 25 μM ; n = 5 ; P = 0 . 001 ) , but not control TAT - GluN2B ( 25 μM ; n = 4 ; P = 0 . 223 ) , decreased native DAPK1 , and this was prevented by NH4Cl ( 20 mM ; n = 5 ; P = 0 . 302 ) . One - way ANOVA , P 0 . 001 , F ( 5 , 24 ) = 13 . 591 . Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control ( white bar ; * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ) or NMDA - treated group ( gray bar ; ΔP 0 . 05 , ΔΔP 0 . 01 and ΔΔΔP 0 . 001 ) . Membranes reprobed for β - actin were used as a loading control . Bars represent relative means ± s . e . m . Sample sizes represent number of individual experiments . Full - length blots are presented in Supplementary Figure 9 .

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

(h) TAT-GluN2BCTM (25 μM; n = 5; P = 0.001), but not control TAT-GluN2B (25 μM; n = 4; P = 0.223), decreased native DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; P = 0.302). One-way ANOVA, P 0.001, F(5,24) = 13.591. Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control (white bar; *P 0.05, **P 0.01 and ***P 0.001) or NMDA-treated group (gray bar; ΔP 0.05, ΔΔP 0.01 and ΔΔΔP 0.001). Membranes reprobed for β-actin were used as a loading control. Bars represent relative means ± s.e.m. Sample sizes represent number of individual experiments. Full-length blots are presented in Supplementary Figure 9.


( a ) Top , the synthetic cell - penetrating α - synuclein targeting peptide TAT - βsynCTM and its control TAT - βsyn . Middle , immunoblots demonstrate that TAT - βsynCTM ( 25 μM ; n = 5 ) , but not the CTM - lacking control peptide TAT - βsyn ( 25 μM ; n = 5 ) , specifically decreased the targeted endogenous α - synuclein ( one - way ANOVA , Tukey post hoc , P 0 . 001 , F ( 3 , 16 ) = 12 . 435 ) , without affecting the level of unrelated control protein PSD - 95 at 4 h ( bottom ) , and this reduction was prevented in the presence of NH4Cl ( 20 mM ; n = 5 ) . Sample sizes represent individual experiments from at least 3 separate primary cultures . (

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

(a) Top, the synthetic cell-penetrating α-synuclein targeting peptide TAT-βsynCTM and its control TAT-βsyn. Middle, immunoblots demonstrate that TAT-βsynCTM (25 μM; n = 5), but not the CTM-lacking control peptide TAT-βsyn (25 μM; n = 5), specifically decreased the targeted endogenous α-synuclein (one-way ANOVA, Tukey post hoc, P 0.001, F(3,16) = 12.435), without affecting the level of unrelated control protein PSD-95 at 4 h (bottom), and this reduction was prevented in the presence of NH4Cl (20 mM; n = 5). Sample sizes represent individual experiments from at least 3 separate primary cultures. (


( b ) Top , PSD - 95 targeting peptide TAT - GluN2B9cCTM and control TAT - GluN2B9c . Middle , TAT - GluN2B9cCTM ( 25 μM ; n = 4 ) , but not Tat - GluN2B9c ( 25 μM ; n = 4 ) , effectively degraded endogenous PSD - 95 ( one - way ANOVA , Tukey post hoc , P 0 . 001 , F ( 3 , 12 ) = 18 . 154 ) without perturbing untargeted protein α - synuclein ( bottom ) . NH4Cl rescued PSD - 95 degradation . Sample sizes represent individual experiments from at least 2 separate primary cultures . Membrane reprobing for β - actin was used as an additional specificity and loading control . ΔP 0 . 05 , * * , ΔΔP 0 . 01 and * * * P 0 . 001 ; bars represent relative mean values ± s . e . m . normalized to the saline control ( arbitrarily set as 1 ) . Full - length blots are presented in Supplementary Figure 9 .

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

(b) Top, PSD-95 targeting peptide TAT-GluN2B9cCTM and control TAT-GluN2B9c. Middle, TAT-GluN2B9cCTM (25 μM; n = 4), but not Tat-GluN2B9c (25 μM; n = 4), effectively degraded endogenous PSD-95 (one-way ANOVA, Tukey post hoc, P 0.001, F(3,12) = 18.154) without perturbing untargeted protein α-synuclein (bottom). NH4Cl rescued PSD-95 degradation. Sample sizes represent individual experiments from at least 2 separate primary cultures. Membrane reprobing for β-actin was used as an additional specificity and loading control. ΔP 0.05, **,ΔΔP 0.01 and ***P 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (arbitrarily set as 1). Full-length blots are presented in Supplementary Figure 9.


( a ) Immunoblotting for phosphorylated DAPK1 ( pDAPK1 ) revealed a time - dependent activation ( dephosphorylation ) of DAPK1 by H2O2 treatment ( 300 μM ; 30 min ; n = 4 ) .

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

(a) Immunoblotting for phosphorylated DAPK1 (pDAPK1) revealed a time-dependent activation (dephosphorylation) of DAPK1 by H2O2 treatment (300 μM; 30 min; n = 4).


( b ) Bath application of 100 μM TAT - GluN2Bct - CTM ( 36 . 54 ± 7 . 1 % of control ; n = 8 ; P = 0 . 001 ) , but not TAT - GluN2Bct ( 89 . 13 ± 10 . 78 % ; n = 7 ; P = 0 . 311 ) , 60 min before and during H2O2 treatment ( 300 μM ; 30 min ) knocked down DAPK1 at 2 h after washout , and this effect was rescued by NH4Cl ( 20 mM ; n = 8 ; * * P = 0 . 003 to control ; P = 0 . 106 to H2O2 - treated ) . One - way ANOVA , F ( 4 , 34 ) = 11 . 628 , P 0 . 001 . Bars represent DAPK1 levels relative to saline control group . β - actin was used as a loading control .

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

(b) Bath application of 100 μM TAT-GluN2Bct-CTM (36.54 ± 7.1% of control; n = 8; P = 0.001), but not TAT-GluN2Bct (89.13 ± 10.78%; n = 7; P = 0.311), 60 min before and during H2O2 treatment (300 μM; 30 min) knocked down DAPK1 at 2 h after washout, and this effect was rescued by NH4Cl (20 mM; n = 8; **P = 0.003 to control; P = 0.106 to H2O2-treated). One-way ANOVA, F(4,34) = 11.628, P 0.001. Bars represent DAPK1 levels relative to saline control group. β-actin was used as a loading control.


( c ) LDH assay revealed that H2O2 treatment ( 300 μM ; 30 min ) resulted in a significant increase in neuronal death 12 h after treatment ( n = 8 ; 2 . 50 ± 0 . 12 ; P 0 . 001 to control ) , which was rescued by breaking down H2O2 with catalase ( 100 U in 10 μl phosphate - buffered saline ; n = 4 ; 1 . 17 ± 0 . 02 ; P = 0 . 001 to H2O2 group ) . H2O2 - induced neurotoxicity was significantly reduced by TAT - GluN2Bct - CTM ( 50 μM ; applied 60 min before and maintained throughout the experiments ; n = 9 ; 1 . 56 ± 0 . 08 ; P = 0 . 001 to H2O2 group ) , but not by TAT - GluN2Bct ( 50 μM ; n = 9 ; 2 . 63 ± 0 . 10 ; P = 0 . 105 to H2O2 group ) or the NMDAR antagonist APV ( 1 μM ; n = 4 ; 2 . 16 ± 0 . 14 ; P = 0 . 169 to H2O2 group ) . NH4Cl abolished the neuroprotective effect of TAT - GluN2Bct - CTM ( n = 4 ; 2 . 39 ± 0 . 27 ; P = 0 . 538 compared to H2O2 group ) . One - way ANOVA , P 0 . 001 , F ( 6 , 41 ) = 26 . 842 . * * * , ΔΔΔP 0 . 001 ; bars represent relative mean values ± s . e . m . normalized to the saline control ( white bar , arbitrarily set as 1 ) . n represents individual experiments from at least 3 separate primary cultures . Full - length blots are presented in Supplementary Figure 9 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 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 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LDH assay revealed that H2O2 treatment (300 μM; 30 min) resulted in a significant increase in neuronal death 12 h after treatment (n = 8; 2.50 ± 0.12; P 0.001 to control), which was rescued by breaking down H2O2 with catalase (100 U in 10 μl phosphate-buffered saline; n = 4; 1.17 ± 0.02; P = 0.001 to H2O2 group). H2O2-induced neurotoxicity was significantly reduced by TAT-GluN2Bct-CTM (50 μM; applied 60 min before and maintained throughout the experiments; n = 9; 1.56 ± 0.08; P = 0.001 to H2O2 group), but not by TAT-GluN2Bct (50 μM; n = 9; 2.63 ± 0.10; P = 0.105 to H2O2 group) or the NMDAR antagonist APV (1 μM; n = 4; 2.16 ± 0.14; P = 0.169 to H2O2 group). NH4Cl abolished the neuroprotective effect of TAT-GluN2Bct-CTM (n = 4; 2.39 ± 0.27; P = 0.538 compared to H2O2 group). One-way ANOVA, P 0.001, F(6,41) = 26.842. ***,ΔΔΔP 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (white bar, arbitrarily set as 1). n represents individual experiments from at least 3 separate primary cultures. Full-length blots are presented in Supplementary Figure 9.


( b ) 2 , 3 , 5 - Triphenyltetrazolium chloride staining of a series of transverse brain sections showed reliable damage in the ipsilateral side following unilateral MCAo . ( c ) Black dashed lines on a section stained as in b represent brain areas removed for immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 2,3,5-Triphenyltetrazolium chloride staining of a series of transverse brain sections showed reliable damage in the ipsilateral side following unilateral MCAo. (c) Black dashed lines on a section stained as in b represent brain areas removed for immunoblotting.


( d ) Immunoblots demonstrated specific DAPK1 knockdown in the infarct ( Ipsi ) but not contralateral ( Contra ) side following application of TAT - GluN2BCTM ( 10 mg per kilogram , i . v . ; n = 3 ; t ( 4 ) = 14 . 459 , P 0 . 001 ) but not TAT - GluN2B ( 10 mg per kilogram , i . v . ; n = 3 ; t ( 4 ) = 0 . 739 , P = 0 . 501 ) . β - actin was used as a loading control ; two - tailed Student ' s t - test * * * P 0 . 001 ; n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots demonstrated specific DAPK1 knockdown in the infarct (Ipsi) but not contralateral (Contra) side following application of TAT-GluN2BCTM (10 mg per kilogram, i.v.; n = 3; t(4) = 14.459, P 0.001) but not TAT-GluN2B (10 mg per kilogram, i.v.; n = 3; t(4) = 0.739, P = 0.501). β-actin was used as a loading control; two-tailed Student's t-test ***P 0.001; n.s., not significant.


( e ) HE ( left ) and immunohistochemical DAPK1 ( right ) staining of adjacent brain sections . Compared with those in saline ( top ) and TAT - GluN2B - treated ( middle ) controls , TAT - GluN2BCTM treatment ( bottom ) selectively reduced infarct area ( left ) and DAPK1 levels ( right ) ipsilaterally . Black outlining delineates infarct areas as visualized with HE staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HE (left) and immunohistochemical DAPK1 (right) staining of adjacent brain sections. Compared with those in saline (top) and TAT-GluN2B-treated (middle) controls, TAT-GluN2BCTM treatment (bottom) selectively reduced infarct area (left) and DAPK1 levels (right) ipsilaterally. Black outlining delineates infarct areas as visualized with HE staining.


( f ) Left , brain sections stained with Fluorojade B in rats injected with saline ( n = 6 ) , TAT - GluN2B ( n = 5 ) or TAT - GluN2BCTM ( n = 5 ) after treatment as shown in a . Right , quantification of cellular damage by counting the number of Fluorojade B - positive cells in each 640 × 640 pixel image at 10 × magnification . TAT - GluN2BCTM ( 10 mg per kilogram ) displayed more prominent neuroprotection in the cortex ( P 0 . 001 ) and striatum ( P 0 . 001 ) as compared to TAT - GluN2B ( 10 mg per kilogram ) . Cortex : H ( 2 ) = 41 . 235 ; P 0 . 001 ; striatum : H ( 2 ) = 38 . 808 ; P 0 . 001 . Kruskal - Wallis ANOVA on ranks with Dunn ' s post hoc ; bars represent relative means ± s . e . m . , * * * , ΔΔΔP 0 . 001 . n values represent tissue from 3 animals collected from at least 2 litters . Scale bars 1 mm in b , c , e , 20 μm in f . Full - length blots are presented in Supplementary Figure 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left, brain sections stained with Fluorojade B in rats injected with saline (n = 6), TAT-GluN2B (n = 5) or TAT-GluN2BCTM (n = 5) after treatment as shown in a. Right, quantification of cellular damage by counting the number of Fluorojade B-positive cells in each 640 × 640 pixel image at 10× magnification. TAT-GluN2BCTM (10 mg per kilogram) displayed more prominent neuroprotection in the cortex (P 0.001) and striatum (P 0.001) as compared to TAT-GluN2B (10 mg per kilogram). Cortex: H(2) = 41.235; P 0.001; striatum: H(2) = 38.808; P 0.001. Kruskal-Wallis ANOVA on ranks with Dunn's post hoc; bars represent relative means ± s.e.m., ***,ΔΔΔP 0.001. n values represent tissue from 3 animals collected from at least 2 litters. Scale bars 1 mm in b,c,e, 20 μm in f. Full-length blots are presented in Supplementary Figure 9.


Same as ( E ) with amiRhen1 - 14 scions and non - transgenic WT or hen1 - 14 rootstocks . Average relative U6 - normalized band - intensity quantifications from two biological replicates are indicated .

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

Same as (E) with amiRhen1-14 scions and non-transgenic WT or hen1-14 rootstocks. Average relative U6-normalized band-intensity quantifications from two biological replicates are indicated.


Phenotype of pSUC2 : : amiRSUL plants in hst - 1 / - 25 / - 26 backgrounds .

Phenotype of pSUC2::amiRSUL plants in hst-1/-25/-26 backgrounds.


RT - qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes . Error - bars : SD . Welch ' s t - test p - values are indicated . n = 5 .

RT-qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes. Error-bars: SD. Welch's t-test p-values are indicated. n=5.


amiRSUL northern analysis in input and AGO1 - immunoprecipitated ( AGO1 - IP ) fractions isolated from leaves of pSUC2 : : amiRSUL plants in the specified genotypes . No Ab : No antibody added to the HST input extract ( IP negative control ) . miR165 / 166 and U6 were probed as endogenous controls . AGO1 western analysis and Coomassie blue ( Coom . ) staining of the western blot membrane are provided as controls . amiRSUL band - intensity quantifications , U6 - normalized for input samples , are indicated .

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

amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated.


amiRSUL northern analysis , in biological duplicates , in scions ( S ) and rootstocks ( R ) in the specified genotypes ' grafting combinations ( amiR : pSUC2 : : amiRSUL ) ( see also Appendix Fig . S12B ) . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated for each 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 O O O O O O O O O O O O O O O O O O O

amiRSUL northern analysis, in biological duplicates, in scions (S) and rootstocks (R) in the specified genotypes' grafting combinations (amiR: pSUC2::amiRSUL) (see also Appendix Fig.S12B). U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated for each tissue.


amiRSUL northern analysis , in biological duplicates , from total RNA extracted from aphids fed on WT or amiR plants , in the specified genetic backgrounds . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated .

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

amiRSUL northern analysis, in biological duplicates, from total RNA extracted from aphids fed on WT or amiR plants, in the specified genetic backgrounds. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.


Localization of HST : GFP and , as a reference , of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves ( C ) or roots ( D ) . CW : calcofluor white staining . Scale bars : 20 µm .

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

Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm.


Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2 : : HST : GFP , pSUC2 : : GFP or pSUC2 : : tmGFP9 . Scale bars : 100 µm . Confocal fluorescence images of the differentiation and division zones of pSUC2 : : GFPhst - 1 or pSUC2 : : tmGFP9hst - 1 root tips . Scale bars : 100 µm .

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

Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2::HST:GFP, pSUC2::GFP or pSUC2::tmGFP9. Scale bars: 100 µm. Confocal fluorescence images of the differentiation and division zones of pSUC2::GFPhst-1 or pSUC2::tmGFP9hst-1 root tips. Scale bars: 100 µm.


Subcellular localizations of HST : GFP expressed from the UBQ10 promoter ( pUBQ ) in Arabidopsis roots ( i ) . Distinct planes of the same root cells imaged by confocal microscopy are shown ( ii - iii ) . PI : propidium iodide staining . Scale bars : 50µm ( i ) ; 10µm ( ii - iii ) .

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

Subcellular localizations of HST:GFP expressed from the UBQ10 promoter (pUBQ) in Arabidopsis roots (i). Distinct planes of the same root cells imaged by confocal microscopy are shown (ii-iii). PI: propidium iodide staining. Scale bars: 50µm (i); 10µm (ii-iii).


Subcellular localizations of hst - 3 : GFP expressed from pUBQ in root cells . Distinct planes of the same root cells imaged by confocal microscopy are shown ( i - ii ) . PI : as in ( A ) . Scale bars : 10µm .

Subcellular localizations of hst-3:GFP expressed from pUBQ in root cells. Distinct planes of the same root cells imaged by confocal microscopy are shown (i-ii). PI: as in (A). Scale bars: 10µm.


miR395 northern analysis in scions ( S ) and hyl1 - 2 rootstocks ( R ) , in the indicated genotypes ' combinations , under SO4 - sufficient ( + SO4 ) or SO4 - starved ( - SO4 ) conditions , in two biological replicates ( rep . ) . U6 was probed as an endogenous control . Relative U6 - normalized band - intensity quantifications are indicated for each tissue in - SO4 conditions .

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

miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions.


miR160 northern analysis in Meselect - separated leaf vasculature and epidermis in WT versus hst - 1 plants , in three biological replicates ( rep . ) . U6 : as in ( A ) . Relative U6 - normalized band - intensity quantifications are indicated .

miR160 northern analysis in Meselect-separated leaf vasculature and epidermis in WT versus hst-1 plants, in three biological replicates (rep.). U6: as in (A). Relative U6-normalized band-intensity quantifications are indicated.


Basic fuchsin staining of protoxylem ( unfilled arrowheads ) and metaxylem ( filled arrowheads ) in WT , shr - 2 or hst - 1 roots . * : protoxylem gap . Scale bars : 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 O O O O O O O O O O O O O

Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm.


( B , Characterization of DONSON ' s chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated for the indicated times in 20 μL of interphase egg extract . Chromatin fractions were isolated and analyzed by immunoblotting . His - p27 ( 100 μg / mL , p27 ( CDKi ) ) , PHA - 767491 ( 50 μM , Cdc7i ) were supplemented as indicated . Similar experiments with ( B ) were performed in triplicate and DONSON signal intensity was quantified by Image 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 B-CONTROLLED_VAR I-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

(B, Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. His-p27 (100 μg/mL, p27 (CDKi)), PHA-767491 (50 μM, Cdc7i) were supplemented as indicated. Similar experiments with (B) were performed in triplicate and DONSON signal intensity was quantified by Image J.


C , Characterization of DONSON ' s chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated for the indicated times in 20 μL of interphase egg extract . Chromatin fractions were isolated and analyzed by immunoblotting . aphidicolin ( 10 μg / mL , aph ) , His - geminin ( 50 μg / mL , gem ) were supplemented as indicated . Similar experiments with ( C ) were performed in triplicate and DONSON signal intensity was quantified by Image 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. aphidicolin (10 μg/mL, aph), His-geminin (50 μg/mL, gem) were supplemented as indicated. Similar experiments with (C) were performed in triplicate and DONSON signal intensity was quantified by Image J.


E ) Characterization of DONSON ' s chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated for the indicated times in 20 μL of interphase egg extract . Chromatin fractions were isolated and analyzed by immunoblotting . NMS - 873 ( 100 μM , p97i ) , and MLN - 4924 ( 20 μM , Culi ) were supplemented as indicated .

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

E) Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. NMS-873 (100 μM, p97i), and MLN-4924 (20 μM, Culi) were supplemented as indicated.


( A ) Immunodepletion from the interphase egg extract was performed using control IgG beads ( mock ) or anti - DONSON antibody beads ( ΔDNS ) . Recombinant Xenopus DONSON ( untagged ) was added to the mock - and DONSON - depleted extracts at 0 μM ( - ) , 0 . 06 μM ( × 1 ) , 0 . 18 ( × 3 ) , and 0 . 36 μM ( × 9 ) as indicated . The extracts ( 1 μL ) were analyzed by immunoblotting , except lanes 9 - 11 , where 0 . 4 ( . 4 ) , 0 . 2 ( . 2 ) , and 0 . 1 ( . 1 ) μL were used . Since the anti - DONSON antibody was recognized by multiple bands , the presumable DONSON band was indicated by dotted lines in a vertically expanded blot . Asterisk ( * ) , non - specific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunodepletion from the interphase egg extract was performed using control IgG beads (mock) or anti-DONSON antibody beads (ΔDNS). Recombinant Xenopus DONSON (untagged) was added to the mock- and DONSON-depleted extracts at 0 μM (-), 0.06 μM (×1), 0.18 (×3), and 0.36 μM (×9) as indicated. The extracts (1 μL) were analyzed by immunoblotting, except lanes 9-11, where 0.4 (.4), 0.2 (.2), and 0.1 (.1) μL were used. Since the anti-DONSON antibody was recognized by multiple bands, the presumable DONSON band was indicated by dotted lines in a vertically expanded blot. Asterisk (*), non-specific band.


( B ) DONSON requirement for the replisome assembly . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of the mock - and DONDON - depleted extracts as prepared in ( A ) for 60 min in the presence of 10 μg / mL aphidicolin . The extracts ( 0 . 5 μL ) and isolated chromatin fractions were analyzed by immunoblotting .

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

(B) DONSON requirement for the replisome assembly. Sperm nuclei (5,000/μL) were incubated in 20 μL of the mock- and DONDON-depleted extracts as prepared in (A) for 60 min in the presence of 10 μg/mL aphidicolin. The extracts (0.5 μL) and isolated chromatin fractions were analyzed by immunoblotting.


( C ) DONSON requirement for replisome assembly . Sperm nuclei were incubated for the indicated times and conditions in the egg extract and the chromatin fractions were analyzed by immunoblotting . Each of two types of anti - DONSON antibodies ( α - DNS # 1 , α - DNS # 2 ) was added at 14 μg / mL to suppress DONSON function .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DONSON requirement for replisome assembly. Sperm nuclei were incubated for the indicated times and conditions in the egg extract and the chromatin fractions were analyzed by immunoblotting. Each of two types of anti-DONSON antibodies (α-DNS #1, α-DNS #2) was added at 14 μg/mL to suppress DONSON function.


( A ) DONSON requirement for DNA replication . Sperm nuclei were incubated for 60 min in egg extract with no IgG ( non ) , control IgG ( α - control ) , or one of the two kinds of anti - DONSON antibodies ( α - DNS # 1 , α - DNS # 2 ) in a final concentration of 14 μg / mL . Cy3 - dCTP was added to all of the reactions at 1 μΜ to visualize the DNA replication . After incubation , the nuclei were fixed , stained with Hoechst 33258 , and observed under a fluorescent microscope . Representative images are shown above . Scale bar ; 50 µm . The relative Cy3 intensity of > 100 nuclei was quantified using Image J and is shown in the box plot below . The boxes define the upper and lower quartiles ; the whiskers define max to min values ; the X - mark indicates each mean value ; the central band indicates each median ; the circles indicates outliers . P - values were calculated using the Mann - Whitney test . ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DONSON requirement for DNA replication. Sperm nuclei were incubated for 60 min in egg extract with no IgG (non), control IgG (α-control), or one of the two kinds of anti-DONSON antibodies (α-DNS #1, α-DNS #2) in a final concentration of 14 μg/mL. Cy3-dCTP was added to all of the reactions at 1 μΜ to visualize the DNA replication. After incubation, the nuclei were fixed, stained with Hoechst 33258, and observed under a fluorescent microscope. Representative images are shown above. Scale bar; 50 µm. The relative Cy3 intensity of >100 nuclei was quantified using Image J and is shown in the box plot below. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.


( B ) DONSON requirement for DNA replication . Sperm nuclei ( 5 , 000 / μL ) were incubated for 60 min in 10 μL of the untreated ( control ) , mock - ( mock ) , and DONDON - depleted ( ΔDNS ) extracts with 2 μM Cy5 - dUTP . Aphidicolin ( aph ) was added to the mock - depleted extract ( 2 . 5 μg / mL ) as a negative control . Various versions of the recombinant Xenopus DONSON ( 1 - 6 ) were added to the DONSON - depleted extracts at about 0 . 06 μM . 1 ; untagged DONSON , 2 and 3 ; GST - DONSON , 4 and 5 ; GST - DONSON ( residues 155 - 579 ) ( ΔN154 ) , 6 ; His - DONSON . In the reactions corresponding to lanes 3 and 5 , 3C protease was added to separate the GST from DONSON . After incubation , the genomic DNA was isolated and subjected to 0 . 8 % TAE agarose gel electrophoresis , followed by SYBR Gold staining . The Cy5 and SYBR Gold fluorescent signals show replicated DNA and total DNA , respectively . The same experiment was performed in triplicate ; the relative Cy5 intensities to the control reaction were quantified using Image J , as shown in the bar graph below . Error bar , mean ± standard deviation ( S . D . ) . P - values were calculated using the unpaired t - test ( two - tailed ) . ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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) DONSON requirement for DNA replication. Sperm nuclei (5,000/μL) were incubated for 60 min in 10 μL of the untreated (control), mock- (mock), and DONDON-depleted (ΔDNS) extracts with 2 μM Cy5-dUTP. Aphidicolin (aph) was added to the mock-depleted extract (2.5 μg/mL) as a negative control. Various versions of the recombinant Xenopus DONSON (1-6) were added to the DONSON-depleted extracts at about 0.06 μM. 1; untagged DONSON, 2 and 3; GST-DONSON, 4 and 5; GST-DONSON (residues 155-579) (ΔN154), 6; His-DONSON. In the reactions corresponding to lanes 3 and 5, 3C protease was added to separate the GST from DONSON. After incubation, the genomic DNA was isolated and subjected to 0.8% TAE agarose gel electrophoresis, followed by SYBR Gold staining. The Cy5 and SYBR Gold fluorescent signals show replicated DNA and total DNA, respectively. The same experiment was performed in triplicate; the relative Cy5 intensities to the control reaction were quantified using Image J, as shown in the bar graph below. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.


( A ) Salt - sensitivity of chromatin - bound DONSON . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of egg extract under each of the conditions as indicated , and the chromatin fractions were isolated with normal buffer plus 0 ( - ) , 0 . 1 , or 0 . 2 M NaCl , and analyzed by immunoblotting . In addition , 0 . 5 μL of each extract ( ext ) was analyzed for comparison .

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

(A) Salt-sensitivity of chromatin-bound DONSON. Sperm nuclei (5,000/μL) were incubated in 20 μL of egg extract under each of the conditions as indicated, and the chromatin fractions were isolated with normal buffer plus 0 (-), 0.1, or 0.2 M NaCl, and analyzed by immunoblotting. In addition, 0.5 μL of each extract (ext) was analyzed for comparison.


( C ) The chromatin fractions were isolated and analyzed by immunoblotting .

(C) The chromatin fractions were isolated and analyzed by immunoblotting.


( E ) ( left ) The isolated genomic DNA was analyzed in the same manner as in Figure 3B . ( right ) The same experiment was repeated six times . The relative Cy5 intensities to the control reaction ( normal α - C ) were quantified using Image J , as shown in the bar graph . Error bar , mean ± standard deviation ( S . D . ) . P - values were calculated using the unpaired t - test ( two - tailed ) . ns , not significant .

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

(E) (left) The isolated genomic DNA was analyzed in the same manner as in Figure 3B. (right) The same experiment was repeated six times. The relative Cy5 intensities to the control reaction (normal α-C) were quantified using Image J, as shown in the bar graph. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.


( A ) Ability of DONSON deletions to support replisome assembly . The DONSON depletion experiment , as that in Figure 2B , was performed using 3 × FLAG - tagged versions of the full length ( amino acid residues 1 - 579 ) or N - terminal / C - terminal deletions ( amino acid residues 1 - 157 , 133 - 579 , 1 - 476 , 1 - 369 ) of Xenopus ( x ) DONSON , whose final concentrations were about 0 . 06 μM . The signals derived from DONSON are indicated with dotted lines . His - p27 and His - geminin were used as negative controls . Asterisk ( * ) , non - specific bands . ( B ) Interaction of DONSON with the replication factors . 3 × FLAG - tagged full length ( xF ) , N - terminal region residue 1 - 157 ( xN ) , middle region residue 133 - 369 ( xM ) , and C - terminal region residue 358 - 579 ( xC ) versions of Xenopus DONSON were added to an interphase egg extract at about 8 . 8 μM . After incubation for 15 min , the whole extract was subjected to a pull - down assay using FLAG - M2 beads , and the precipitated proteins were analyzed by immunoblotting ( FLAG - IP ) . The extracts ( 2 . 5 % ) were analyzed for comparison ( Input ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ability of DONSON deletions to support replisome assembly. The DONSON depletion experiment, as that in Figure 2B, was performed using 3×FLAG-tagged versions of the full length (amino acid residues 1-579) or N-terminal/C-terminal deletions (amino acid residues 1-157, 133-579, 1-476, 1-369) of Xenopus (x) DONSON, whose final concentrations were about 0.06 μM. The signals derived from DONSON are indicated with dotted lines. His-p27 and His-geminin were used as negative controls. Asterisk (*), non-specific bands. (B) Interaction of DONSON with the replication factors. 3×FLAG-tagged full length (xF), N-terminal region residue 1-157 (xN), middle region residue 133-369 (xM), and C-terminal region residue 358-579 (xC) versions of Xenopus DONSON were added to an interphase egg extract at about 8.8 μM. After incubation for 15 min, the whole extract was subjected to a pull-down assay using FLAG-M2 beads, and the precipitated proteins were analyzed by immunoblotting (FLAG-IP). The extracts (2.5%) were analyzed for comparison (Input).


( D ) Identification of GINS - and Polε - interacting motifs in the N - terminal region of DONSON . The FLAG - IP experiment , as in ( B ) , was performed with 2 μM of 3 × FLAG - tagged versions of wild - type ( xN - WT ) , P6A / Y8A ( xN - 2A ) , N67A / P68A / F69A ( xN - 3A ) , and P6A / Y8A / N67A / P68A / F69A ( xN - 5A ) of Xenopus DONSON each with an N - terminal with 157 amino acids . The extracts ( 6 . 7 % ) were analyzed for comparison ( Input ) . ( E ) Interaction of human DONSON with Xenopus GINS and Polε in egg extract . The FLAG - IP experiment , as in ( B ) , was performed with 1 μM of 3 × FLAG - tagged versions of wild - type ( hF - WT ) , P6A / Y8A ( hF - 2A ) , N78A / P79A / F80A ( hF - 3A ) , P6A / Y8A / N78A / P79A / F80A ( hF - 5A ) , K489T ( hF - KT ) of full length human DONSON each as well as 3 × FLAG - tagged wild - type Xenopus DONSON ( xF - WT ) . The extracts ( 5 % ) were analyzed for comparison ( Input ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Identification of GINS- and Polε-interacting motifs in the N-terminal region of DONSON. The FLAG-IP experiment, as in (B), was performed with 2 μM of 3×FLAG-tagged versions of wild-type (xN-WT), P6A/Y8A (xN-2A), N67A/P68A/F69A (xN-3A), and P6A/Y8A/N67A/P68A/F69A (xN-5A) of Xenopus DONSON each with an N-terminal with 157 amino acids. The extracts (6.7%) were analyzed for comparison (Input). (E) Interaction of human DONSON with Xenopus GINS and Polε in egg extract. The FLAG-IP experiment, as in (B), was performed with 1 μM of 3×FLAG-tagged versions of wild-type (hF-WT), P6A/Y8A (hF-2A), N78A/P79A/F80A (hF-3A), P6A/Y8A/N78A/P79A/F80A (hF-5A), K489T (hF-KT) of full length human DONSON each as well as 3×FLAG-tagged wild-type Xenopus DONSON (xF-WT). The extracts (5%) were analyzed for comparison (Input).


( A ) Requirement of GINS - and Polε - interacting motifs within DONSON for replisome assembly . This DONSON depletion experiment , as that in Figure 2B , was performed using the same recombinant proteins as in Figure 5E . Human proteins were added at 0 . 3 μM each and Xenopus protein at 0 . 06 μM . His - p27 ( p27 ) and His - geminin ( gem ) were used as negative controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Requirement of GINS- and Polε-interacting motifs within DONSON for replisome assembly. This DONSON depletion experiment, as that in Figure 2B, was performed using the same recombinant proteins as in Figure 5E. Human proteins were added at 0.3 μM each and Xenopus protein at 0.06 μM. His-p27 (p27) and His-geminin (gem) were used as negative controls.


( B ) Requirement of GINS - and Polε - interacting motifs within DONSON for replication activity . The same experiment procedure was used as in Figure 3A for the mock - and DONSON - depleted extracts with the same recombinant proteins as in Figure 5E . Xenopus proteins were added at 0 . 06 μM each while human proteins were added at 0 . 3 μM . The relative Cy3 intensity of > 100 nuclei was quantified using Image J and is shown in the box plot . The boxes define the upper and lower quartiles ; the whiskers define max to min values ; the X - mark indicates each mean value ; the central band indicates each median ; the circles indicates outliers . P - values were calculated using the Mann - Whitney test . ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Requirement of GINS- and Polε-interacting motifs within DONSON for replication activity. The same experiment procedure was used as in Figure 3A for the mock- and DONSON-depleted extracts with the same recombinant proteins as in Figure 5E. Xenopus proteins were added at 0.06 μM each while human proteins were added at 0.3 μM. The relative Cy3 intensity of >100 nuclei was quantified using Image J and is shown in the box plot. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.


( A , Requirement of GINS , Cdc45 for the DONSON chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of mock - , GINS - , Cdc45 - depleted egg extract ΔGINS , ΔCdc45 for the indicated times and conditions , and the chromatin fractions were isolated and analyzed by immunoblotting . The extracts ( 0 . 5 μL ) were also analyzed . Replication proteins were categorized into three groups ( Group - 1 , - 2 , - 3 ) based on their dependency on GINS ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Requirement of GINS, Cdc45 for the DONSON chromatin association. Sperm nuclei (5,000/μL) were incubated in 20 μL of mock-, GINS-, Cdc45- depleted egg extract ΔGINS, ΔCdc45 for the indicated times and conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. The extracts (0.5 μL) were also analyzed. Replication proteins were categorized into three groups (Group-1, -2, -3) based on their dependency on GINS, Cdc45


B ) Requirement of TopBP1 for the DONSON chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of mock - , or TopBP1 - depleted egg extract , ΔTopBP1 ) for the indicated times and conditions , and the chromatin fractions were isolated and analyzed by immunoblotting . The extracts ( 0 . 5 μL ) were also analyzed . Replication proteins were categorized into three groups ( Group - 1 , - 2 , - 3 ) based on their dependency on TopBP1 .

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B) Requirement of TopBP1 for the DONSON chromatin association. Sperm nuclei (5,000/μL) were incubated in 20 μL of mock-, or TopBP1- depleted egg extract , ΔTopBP1) for the indicated times and conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. The extracts (0.5 μL) were also analyzed. Replication proteins were categorized into three groups (Group-1, -2, -3) based on their dependency on TopBP1.


( C ) ( above ) Requirement of DONSON for CDK and DDK function in the replisome assembly . Sperm nuclei ( 5 , 000 / μL ) were incubated for 50 min in 120 μL of egg extract with control IgG ( α - cont ) or anti - DONSON antibody # 1 ( α - DNS ) . Each part of this first reaction ( 1st ) was divided equally into six parts , and the nuclear fractions were isolated and transferred to 20 μL of flesh extract with control IgG ( α - cont ) or anti - DONSON antibody # 1 ( α - DNS ) in the presence or absence of CDK inhibitors ( Ci : 100 μM roscovitine , 100 μg / mL His - p27 ) and Cdc7 inhibitor ( 7i : 50 μM PHA - 767491 ) . Aphidicolin was added to the first and second extracts at 10 μg / mL to suppress replication termination . After incubation for 40 min in the second reaction ( 2nd ) , the chromatin fractions were isolated and analyzed by immunoblotting . In addition , the extracts ( 0 . 5 μL ) were analyzed ( ext ) . ( below ) It is summarized whether or not replisome assembly was allowed under each condition , where DONSON , CDK , and Cdc7 were either functional ( + ) or non - functional ( - ) in the 1st and 2nd incubations .

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

(C) (above) Requirement of DONSON for CDK and DDK function in the replisome assembly. Sperm nuclei (5,000/μL) were incubated for 50 min in 120 μL of egg extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS). Each part of this first reaction (1st) was divided equally into six parts, and the nuclear fractions were isolated and transferred to 20 μL of flesh extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS) in the presence or absence of CDK inhibitors (Ci: 100 μM roscovitine, 100 μg/mL His-p27) and Cdc7 inhibitor (7i: 50 μM PHA-767491). Aphidicolin was added to the first and second extracts at 10 μg/mL to suppress replication termination. After incubation for 40 min in the second reaction (2nd), the chromatin fractions were isolated and analyzed by immunoblotting. In addition, the extracts (0.5 μL) were analyzed (ext). (below) It is summarized whether or not replisome assembly was allowed under each condition, where DONSON, CDK, and Cdc7 were either functional (+) or non-functional (-) in the 1st and 2nd incubations.


A Representative images show expression of YAP in normal cervical tissues , early - stage cervical cancer ( stage I II ) , and advanced - stage cervical cancer ( stage III IV ) . Note the significant increase in YAP immunosignals ( brown staining ) in the advanced - stage tumors . Tissue core size : 1 . 5 mm . B Positivity ( percentage of positively stained cells relative to the total number of cells in the tissue section ) of YAP immunosignals in the normal and cancerous tissues . Data were analyzed with unpaired t - test in GraphPad Prism 5 with Welch ' s correction . Bars represent means ± SEM ( n = 10 for normal tissues ; n = 69 for tumor tissues ; * * * P 0 . 0001 ) .

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A Representative images show expression of YAP in normal cervical tissues, early-stage cervical cancer (stage I II), and advanced-stage cervical cancer (stage III IV). Note the significant increase in YAP immunosignals (brown staining) in the advanced-stage tumors. Tissue core size: 1.5 mm.B Positivity (percentage of positively stained cells relative to the total number of cells in the tissue section) of YAP immunosignals in the normal and cancerous tissues. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Bars represent means ± SEM (n = 10 for normal tissues; n = 69 for tumor tissues; ***P 0.0001).


C A representative image showing the expression of YAP ( brown staining ) in normal cervical tissue . Scale bar = 20 μm . D - F Representative images showing the expression of YAP in ( D ) early - stage cervical cancer tissue ( stage Ib , T1N0M0 ) ; ( E ) medium - stage cervical cancer tissue ( stage IIb , T2N0M0 ) ; and ( F ) advanced - stage cervical cancer tissue ( stage IIIb , T3N1M0 ) . Scale bar = 20 μm . G , H High - resolution images showing the cellular location of YAP in ( G ) early - stage cervical cancer tissue and ( H ) advanced - stage cervical cancer tissue . Scale bar = 20 μm .

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C A representative image showing the expression of YAP (brown staining) in normal cervical tissue. Scale bar = 20 μm.D-F Representative images showing the expression of YAP in (D) early-stage cervical cancer tissue (stage Ib, T1N0M0); (E) medium-stage cervical cancer tissue (stage IIb, T2N0M0); and (F) advanced-stage cervical cancer tissue (stage IIIb, T3N1M0). Scale bar = 20 μm.G, H High-resolution images showing the cellular location of YAP in (G) early-stage cervical cancer tissue and (H) advanced-stage cervical cancer tissue. Scale bar = 20 μm.


I Multidimensional cancer genomics data analysis results showing cross - cancer YAP gene alteration . The histogram showed the frequencies of YAP gene mutation , deletion , and amplification across cancers . Data were extracted from TCGA database and analyzed using cBioPortal online analyzing tools . The results indicate that the highest alteration frequency of YAP gene occurs in cervical cancer .

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I Multidimensional cancer genomics data analysis results showing cross-cancer YAP gene alteration. The histogram showed the frequencies of YAP gene mutation, deletion, and amplification across cancers. Data were extracted from TCGA database and analyzed using cBioPortal online analyzing tools. The results indicate that the highest alteration frequency of YAP gene occurs in cervical cancer.


A , C , E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ ME180 - MXIV ( control ) , ME180 - YAP , and ME180 - YAPS127A cells ] ( A ) ; HT3 cell lines [ HT3 - MXIV ( control ) , HT3 - YAP , and HT3 - YAPS127A cells ] ( C ) ; and Ect1 cell lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , and Ect1 - YAPS127A cells ] ( E ) . β - Actin was used as a protein loading control .

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A, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.


B , D , F Growth curves of YAP - overexpressed ME180 cell lines [ ME180 - MXIV ( control ) , ME180 - YAP , and ME180 - YAPS127A cells ] ( B ) ; HT3 cell lines [ HT3 - MXIV ( control ) , HT3 - YAP , and HT3 - YAPS127A cells ] ( D ) ; and Ect1 cell lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , and Ect1 - YAPS127A cells ] ( F ) . Each point represents the mean ± SEM ( n = 4 ) . * * * P < 0 . 0001 , ME180 - MXIV vs . ME180 - YAP cells and ME180 - MXIV vs . ME180 - YAPS127A cells on day 8 . * * * P < 0 . 0001 , HT3 - MXIV vs . HT3 - YAP cells and HT3 - MXIV vs . HT3 - YAPS127A cells on day 8 . * * * P < 0 . 0001 , Ect1 - MXIV vs . Ect1 - Ect1 - YAPS127A cells on day 4 . * P < 0 . 0074 , Ect1 - MXIV vs . Ect1 - YAP cells on day 4 .

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B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.


G Western blot showing YAP levels in non - targeting control siRNA ( siCtrl ) - and YAP siRNA ( siYAP ) - transfected HT3 cells .

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G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.


H Proliferation of HT3 cells treated with control ( siCtrl ) or YAP siRNA ( siYAP ) . Each point represents the mean ± SEM ( n = 5 ) . * * * P < 0 . 001 compared with siCtrl ( siCtrl vs . siYAP , P = 0 . 0002 ) .

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H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).


A Representative images showing colonies formed by Ect1 - MXIV , Ect1 - YAP , and Ect1 - YAPS127A cells after growth in soft agar for 9 days . Scale bar : 500 μm . B Quantitative data showing colony formation in Ect1 - MXIV , Ect1 - YAP , and Ect1 - YAPS127A cells . Each bar represents mean ± SEM of five independent experiments . Bars with different letters are significantly different from each other ( Ect1 - MXIV vs . Ect1 - YAP , P = 0 . 0003 ; Ect1 - MXIV vs . Ect1 - YAPS127A , P = 0 . 0002 ) .

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A Representative images showing colonies formed by Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells after growth in soft agar for 9 days. Scale bar: 500 μm.B Quantitative data showing colony formation in Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells. Each bar represents mean ± SEM of five independent experiments. Bars with different letters are significantly different from each other (Ect1-MXIV vs. Ect1-YAP, P = 0.0003; Ect1-MXIV vs. Ect1-YAPS127A, P = 0.0002).


C , E Representative images showing the anchorage - independent growth of YAP - expressing HT3 ( C ) and ME180 ( E ) cervical cancer cell lines . Anchorage - independent cell growth was determined by the soft agar colony formation assay . Scale bar : 500 μm . D , F Fluorescence - based quantitative analysis showing the differences of anchorage - independent growth in HT3 - MXIV , HT3 - YAP , and HT3 - YAPS127A ( D ) and ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells ( F ) . The anchorage - independent cell growth was determined using a CytoSelect 96 - Well Cell Transformation Assay kit , and data are presented as relative fluorescent units ( RFU ) . Each bar represents mean ± SEM of four independent experiments . Bars with different letters are significantly different from each other ( HT3 - MXIV vs . HT3 - YAP , P = 0 . 0002 ; HT3 - MXIV vs . HT3 - YAPS127A , P < 0 . 0001 ; ME - MXIV vs . ME - YAP , P = 0 . 0011 ; ME - MXIV vs . ME - YAPS127A , P = 0 . 0013 ) .

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C, E Representative images showing the anchorage-independent growth of YAP-expressing HT3 (C) and ME180 (E) cervical cancer cell lines. Anchorage-independent cell growth was determined by the soft agar colony formation assay. Scale bar: 500 μm.D, F Fluorescence-based quantitative analysis showing the differences of anchorage-independent growth in HT3-MXIV, HT3-YAP, and HT3-YAPS127A (D) and ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells (F). The anchorage-independent cell growth was determined using a CytoSelect 96-Well Cell Transformation Assay kit, and data are presented as relative fluorescent units (RFU). Each bar represents mean ± SEM of four independent experiments. Bars with different letters are significantly different from each other (HT3-MXIV vs. HT3-YAP, P = 0.0002; HT3-MXIV vs. HT3-YAPS127A, P < 0.0001; ME-MXIV vs. ME-YAP, P = 0.0011; ME-MXIV vs. ME-YAPS127A, P = 0.0013).


A , B Representative images of tumorxenografts of ME180 - MXIV ( MX , right flank ) , ME180 - YAP ( A ) ( YAP , left flank ) , and ME180 - YAPS127A ( B ) ( YAPS127A , left flank ) cells implanted in athymic nude mice . Red lines indicate the edge of tumors . C The growth curve of humantumorxenografts derived from control ( ME - MX ) and YAP - overexpressing ME180cervical cancer cell lines ( ME - YAP , ME - YAPS127A ) implanted in the athymic nude mice . Each point represents mean ± SEM of six tumors ( control n = 10 ) .

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A, B Representative images of tumorxenografts of ME180-MXIV (MX, right flank), ME180-YAP (A) (YAP, left flank), and ME180-YAPS127A (B) (YAPS127A, left flank) cells implanted in athymic nude mice. Red lines indicate the edge of tumors.C The growth curve of humantumorxenografts derived from control (ME-MX) and YAP-overexpressing ME180cervical cancer cell lines (ME-YAP, ME-YAPS127A) implanted in the athymic nude mice. Each point represents mean ± SEM of six tumors (control n = 10).


D Representative images showing the relative size of tumors from control and YAP - overexpressing ME180 cervical cancer cells . E The average weight of tumor xenografts from the control and YAP - overexpressing ME180 cell lines . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc tests . Each bar represents the mean ± SEM ( n = 10 for control , n = 6 for YAP and YAPS127A groups ) . Bars with different letters are significantly different from each other ( MXIV vs . YAP , P = 0 . 0008 ; MXIV vs . YAPS127A , P < 0 . 0001 ) .

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D Representative images showing the relative size of tumors from control and YAP-overexpressing ME180 cervical cancer cells.E The average weight of tumor xenografts from the control and YAP-overexpressing ME180 cell lines. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents the mean ± SEM (n = 10 for control, n = 6 for YAP and YAPS127A groups). Bars with different letters are significantly different from each other (MXIV vs. YAP, P = 0.0008; MXIV vs. YAPS127A, P < 0.0001).


F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells .

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F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells.


G Expression of Ki67 in the tumor xenografts of ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells implanted in athymic nude mice . Ki67 was visualized using an Alexa - 488 ( green ) - conjugated secondary antibody . Actin was visualized using an Alexa - 594 ( red ) - conjugated secondary antibody . Nuclei were stained with DAPI . Scale bar : 20 μm . Note the significant increase in the Ki67 - positive cells in the ME180 - YAP and ME180 - YAPS127A tumor xenografts .

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G Expression of Ki67 in the tumor xenografts of ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells implanted in athymic nude mice. Ki67 was visualized using an Alexa-488 (green)-conjugated secondary antibody. Actin was visualized using an Alexa-594 (red)-conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 20 μm. Note the significant increase in the Ki67-positive cells in the ME180-YAP and ME180-YAPS127A tumor xenografts.


H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector ( shCtrl ) or lentivirus - based YAP shRNAs ( shYAP # 1 or shYAP # 2 ) .

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H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector (shCtrl) or lentivirus-based YAP shRNAs (shYAP#1 or shYAP#2).


I Representative images showing tumor xenografts derived from ME180 - shCtrl cells ( left flank ) and ME180 - shYAP # 1 cells ( right flank ) ( n = 6 ) .

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I Representative images showing tumor xenografts derived from ME180-shCtrl cells (left flank) and ME180-shYAP#1 cells (right flank) (n = 6).


J Representative images showing the relative size of tumors derived from ME180 cervical cancer cells transfected with control shRNA ( left , shCtrl ) or YAP shRNA # 1 ( right , shYAP # 1 ) ( n = 6 ) .

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J Representative images showing the relative size of tumors derived from ME180 cervical cancer cells transfected with control shRNA (left, shCtrl) or YAP shRNA#1 (right, shYAP#1) (n = 6).


RT - PCR results showing that YAP stimulates the mRNA expressions of EGFR , AREG , and TGF - α in ME180 cervical cancer cell . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc tests . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( YAP mRNA : MXIV vs . YAP , P = 0 . 0003 ; MXIV vs . YAPS127A , P = 0 . 0001 ; EGFR mRNA : MXIV vs . YAP , P = 0 . 0066 ; MXIV vs . YAPS127A , P = 0 . 0003 ; TGF - α mRNA : MXIV vs . YAP , P = 0 . 0235 ; MXIV vs . YAPS127A , P = 0 . 0040 ; AREG mRNA : MXIV vs . YAP , P = 0 . 0002 ; MXIV vs . YAPS127A , P < 0 . 0001 ) .

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RT-PCR results showing that YAP stimulates the mRNA expressions of EGFR, AREG, and TGF-α in ME180 cervical cancer cell. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (YAP mRNA: MXIV vs. YAP, P = 0.0003; MXIV vs. YAPS127A, P = 0.0001; EGFR mRNA: MXIV vs. YAP, P = 0.0066; MXIV vs. YAPS127A, P = 0.0003; TGF-α mRNA: MXIV vs. YAP, P = 0.0235; MXIV vs. YAPS127A, P = 0.0040; AREG mRNA: MXIV vs. YAP, P = 0.0002; MXIV vs. YAPS127A, P < 0.0001).


Proliferation of ME180 cells incubated in medium containing 1 % FBS in the absence ( control ) or presence of 10 ng / ml TGF - α . Data were analyzed with unpaired t - test in GraphPad Prism 5 with Welch ' s correction . Each point represents the mean ± SEM of four independent repeats . * * * P < 0 . 0001 versus control on day 5 .

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Proliferation of ME180 cells incubated in medium containing 1% FBS in the absence (control) or presence of 10 ng/ml TGF-α. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Each point represents the mean ± SEM of four independent repeats. ***P < 0.0001 versus control on day 5.


TGF - α treatment ( 10 ng / ml , 24 h ) promotes ME180 cell cycle progression . G1 , S , and G2 indicate cells in G1 phase , DNA synthesis phase , and the G2 / M phase , respectively , of cell cycle . Apo : apoptotic cells .

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TGF-α treatment (10 ng/ml, 24 h) promotes ME180 cell cycle progression. G1, S, and G2 indicate cells in G1 phase, DNA synthesis phase, and the G2/M phase, respectively, of cell cycle. Apo: apoptotic cells.


Representative images showing the morphology of ME180 cells with or without TGF - α ( 10 ng / ml ) treatment for 24 h ( scale bar : 50 μm ) or 108 h ( scale bar : 25 μm ) . Please note the elongation of ME180 cells after TGF - α treatment for 24 h ( TGF - α , 24 h ) and the formation of multiple layers in ME180 cells after TGF - α treatment for 108 h ( TGF - α , 108 h ) .

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Representative images showing the morphology of ME180 cells with or without TGF-α (10 ng/ml) treatment for 24 h (scale bar: 50 μm) or 108 h (scale bar: 25 μm). Please note the elongation of ME180 cells after TGF-α treatment for 24 h (TGF-α, 24 h) and the formation of multiple layers in ME180 cells after TGF-α treatment for 108 h (TGF-α, 108 h).


Effect of TGF - α on the migration of ME180 cells . TGF - α treatment ( 100 ng / ml , 10 h ) drastically stimulated the migration of ME180 cells .

Effect of TGF-α on the migration of ME180 cells. TGF-α treatment (100 ng/ml, 10 h) drastically stimulated the migration of ME180 cells.


Western blot analysis showing effects of TGF - α on phosphorylation of EGFR , YAP , AKT , and ERK1 / 2 . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with TGF - α ( 100 ng / ml ) for 0 , 5 , 10 , 30 , 60 or 120 min .

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Western blot analysis showing effects of TGF-α on phosphorylation of EGFR, YAP, AKT, and ERK1/2. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 5, 10, 30, 60 or 120 min.


Western blot analysis showing effects of TGF - α on the phosphorylation of the components of the Hippo signaling pathway . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with TGF - α ( 100 ng / ml ) for 0 , 30 , 60 min .

Western blot analysis showing effects of TGF-α on the phosphorylation of the components of the Hippo signaling pathway. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 30, 60 min.


Real - time PCR determines the action of TGF - α and YAP levels on the mRNA expression of AREG . Each bar represents the mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( Ctrl vs . TGF - α , P < 0 . 0001 ; MXIV vs . YAP , P < 0 . 0001 ; MXIV vs . YAPS127A , P < 0 . 0001 ) .

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Real-time PCR determines the action of TGF-α and YAP levels on the mRNA expression of AREG. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (Ctrl vs. TGF-α, P < 0.0001; MXIV vs. YAP, P < 0.0001; MXIV vs. YAPS127A, P < 0.0001).


Real - time PCR analysis showing that knockdown of YAP with YAP siRNA ( siYAP ) significantly suppressed TGF - α - induced ( FBS 10 % , TGF - α : 10 ng / ml for 48 h ) AREG expression in ME180 cells . siCTRL , a non - targeting siRNA , was used as a negative control . Each bar represents the mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0067 ) .

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Real-time PCR analysis showing that knockdown of YAP with YAP siRNA (siYAP) significantly suppressed TGF-α-induced (FBS 10%, TGF-α: 10 ng/ml for 48 h) AREG expression in ME180 cells. siCTRL, a non-targeting siRNA, was used as a negative control. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0067).


Proliferation of ME180 cells ( FBS 1 % ) treated with control ( siCTRL ) or YAP ( siYAP ) prior to treatment with control medium or TGF - α ( 10 ng / ml ) for 108 h . Each bar represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P = 0 . 0058 ; siYAP + CTRL vs . siYAP + TGF - α , P = 0 . 1840 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0013 ) .

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Proliferation of ME180 cells (FBS 1%) treated with control (siCTRL) or YAP (siYAP) prior to treatment with control medium or TGF-α (10 ng/ml) for 108 h. Each bar represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P = 0.0058; siYAP+CTRL vs. siYAP+TGF-α, P = 0.1840; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0013).


Quantitative data of the wound - healing assay showing the migration of ME180 cells that were treated with control ( siCTRL ) or YAP siRNA ( siYAP ) prior to treatment with or without TGF - α for 12 h . Each bar in bar graphs represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0041 ) .

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Quantitative data of the wound-healing assay showing the migration of ME180 cells that were treated with control (siCTRL) or YAP siRNA (siYAP) prior to treatment with or without TGF-α for 12 h. Each bar in bar graphs represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0041).


Western blot analysis showing that AREG treatment induced dephosphorylation of LATS1 , MOB1 , and YAP . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with AREG ( 50 ng / ml ) for 0 , 30 , 60 min .

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Western blot analysis showing that AREG treatment induced dephosphorylation of LATS1, MOB1, and YAP. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with AREG (50 ng/ml) for 0, 30, 60 min.


AREG ( 50 ng / ml ) treatment induced appearance of elongated cells in ME180 cells ( top and middle panels ) and significantly stimulated ME180 cell proliferation ( lower panel ) . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 003 ) . Scale bar : 100 μm .

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AREG (50 ng/ml) treatment induced appearance of elongated cells in ME180 cells (top and middle panels) and significantly stimulated ME180 cell proliferation (lower panel). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.003). Scale bar: 100 μm.


Wound - healing assay showing that AREG stimulates migration of ME180 cervical cancer cells within 10 h in serum - free medium . Scale bar : 200 μm .

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Wound-healing assay showing that AREG stimulates migration of ME180 cervical cancer cells within 10 h in serum-free medium. Scale bar: 200 μm.


Real - time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression . Each bar represents mean ± SEM ( n = 9 ) . Bars with different letters are significantly different from each other ( P < 0 . 0001 ) .

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Real-time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression. Each bar represents mean ± SEM (n = 9). Bars with different letters are significantly different from each other (P < 0.0001).


Real - time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA ( siYAP ) significantly suppressed AREG - induced AREG mRNA expression . siCTRL ( non - target siRNA ) was used as a siRNA control . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + AREG , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + AREG , P < 0 . 0001 ; siCTRL + AREG vs . siYAP + AREG , P = 0 . 0037 ) .

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Real-time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA (siYAP) significantly suppressed AREG-induced AREG mRNA expression. siCTRL (non-target siRNA) was used as a siRNA control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+AREG, P < 0.0001; siYAP+CTRL vs. siYAP+AREG, P < 0.0001; siCTRL+AREG vs. siYAP+AREG, P = 0.0037).


Representative images showing the morphology of spheroids derived from ME180 - siCtrl and ME180 - siLATS1 / 2 cells growing in a 3D hanging - drop culture system for 10 days . Scale bar : 1 . 0 mm .

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Representative images showing the morphology of spheroids derived from ME180-siCtrl and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system for 10 days. Scale bar: 1.0 mm.


Quantitative data showing changes in the volume of spheroids derived from ME180 - siCTRL and ME180 - siLATS1 / 2 cells growing in a 3D hanging - drop culture system . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0004 ) .

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Quantitative data showing changes in the volume of spheroids derived from ME180-siCTRL and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0004).


Quantitative data showing changes in the volume of spheroids derived from ME180 cells growing in a 3D hanging - drop culture system in the absence or presence of AREG ( 20 ng / ml , 8 days ) . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0030 ) .

Quantitative data showing changes in the volume of spheroids derived from ME180 cells growing in a 3D hanging-drop culture system in the absence or presence of AREG (20 ng/ml, 8 days). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0030).


Soft agar assay showing the effect of AG1478 and VTPF on colony formation in ME180 - MXIV , ME180 - YAP and ME180 - YAPS127A cells . Scale bar : 500 μm .

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Soft agar assay showing the effect of AG1478 and VTPF on colony formation in ME180-MXIV, ME180-YAP and ME180-YAPS127A cells. Scale bar: 500 μm.


Representative images showing the effect of AREG , EGFR inhibitor ( AG1478 ) , and YAP antagonist verteporfin ( VTPF ) on the growth of ME180 cell in a 3D hanging - drop culture system . ME180 cells were incubated in the 3D hanging - drop culture system for 10 days in the absence or presence of AREG , AG1478 or verteporfin for 8 days . Scale bar : 1 . 0 mm .

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Representative images showing the effect of AREG, EGFR inhibitor (AG1478), and YAP antagonist verteporfin (VTPF) on the growth of ME180 cell in a 3D hanging-drop culture system. ME180 cells were incubated in the 3D hanging-drop culture system for 10 days in the absence or presence of AREG, AG1478 or verteporfin for 8 days. Scale bar: 1.0 mm.


Effect of recombinant HPV16 E6 protein on the proliferation of HT3 cells . Each point represents mean ± SEM ( n = 5 ) . * * * P = 0 . 0001 compared with control ( Ctrl ) on day 4 . HT3 cells were cultured in serum - reduced medium in the presence or absence of recombinant HPV16 E6 ( 400 nM ) .

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Effect of recombinant HPV16 E6 protein on the proliferation of HT3 cells. Each point represents mean ± SEM (n = 5). ***P = 0.0001 compared with control (Ctrl) on day 4. HT3 cells were cultured in serum-reduced medium in the presence or absence of recombinant HPV16 E6 (400 nM).


Recombinant HPV16 E6 protein increased YAP protein level , but had no effect on β - actin protein level in HT3 cells . Cell culture and treatment procedure are the same as described in ( A ) .

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Recombinant HPV16 E6 protein increased YAP protein level, but had no effect on β-actin protein level in HT3 cells. Cell culture and treatment procedure are the same as described in (A).


AREG mRNA levels in HT3 cells incubated for 48 h with or without recombinant HPV E6 . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0042 ) .

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AREG mRNA levels in HT3 cells incubated for 48 h with or without recombinant HPV E6. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0042).


Western blotting analysis showing the effect of HPV16E6 ( 400 nM , 48 h ) on YAP protein levels in HT3 cells transfected with non - targeting control siRNA ( siCtrl ) or YAP siRNA ( siYAP ) .

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Western blotting analysis showing the effect of HPV16E6 (400 nM, 48 h) on YAP protein levels in HT3 cells transfected with non-targeting control siRNA (siCtrl) or YAP siRNA (siYAP).


Effect of YAP on HPV16 E6 stimulation of HT3 cell proliferation . siCtrl : non - targeting control siRNA ; siYAP : YAP siRNA ; E6 : 400 nM recombinant HPV16 E6 , 48 h . Each bar represents mean ± SEM ( n = 4 ) . Bars with the same letters are not significantly different from each other ( siCtrl vs . siCtrl + E6 , P = 0 . 0232 ; siCtrl + E6 vs . siYAP + E6 , P = 0 . 0011 ) .

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Effect of YAP on HPV16 E6 stimulation of HT3 cell proliferation. siCtrl: non-targeting control siRNA; siYAP: YAP siRNA; E6: 400 nM recombinant HPV16 E6, 48 h. Each bar represents mean ± SEM (n = 4). Bars with the same letters are not significantly different from each other (siCtrl vs. siCtrl+E6, P = 0.0232; siCtrl+E6 vs. siYAP+E6, P = 0.0011).


Knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA ( siE6 ) reduced YAP protein in HeLa cells , while knockdown of YAP with YAP siRNA ( siYAP ) in these cells had no effect on the mRNA level of HPV18 E6 . siCtrl : non - targeting control siRNA .

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Knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA (siE6) reduced YAP protein in HeLa cells, while knockdown of YAP with YAP siRNA (siYAP) in these cells had no effect on the mRNA level of HPV18 E6. siCtrl: non-targeting control siRNA.


Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6 - specific siRNA ( siE6 ) significantly suppressed mRNA expression of AREG . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siHPV18E6 , P = 0 . 0181 ; siCtrl vs . siYAP , P = 0 . 0005 ) .

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Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6-specific siRNA (siE6) significantly suppressed mRNA expression of AREG. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siHPV18E6, P = 0.0181; siCtrl vs. siYAP, P = 0.0005).


Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6 - specific siRNA ( siE6 ) significantly suppressed cell growth ( n = 9 , siCtrl vs . siHPV18E6 , P = 0 . 0002 ; siCtrl vs . siYAP , P = 0 . 0004 ) .

Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6-specific siRNA (siE6) significantly suppressed cell growth (n = 9, siCtrl vs. siHPV18E6, P = 0.0002; siCtrl vs. siYAP, P = 0.0004).


Concentrations of AREG in the culture medium of HeLa cells transfected with non - targeting control siRNA ( siCTRL ) or HPV18 E6 siRNA ( siHPV18E6 ) . Each bar represents mean ± SEM ( n = 6 ) . Bars with different letters are significantly different from each other ( P = 0 . 0461 ) .

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Concentrations of AREG in the culture medium of HeLa cells transfected with non-targeting control siRNA (siCTRL) or HPV18 E6 siRNA (siHPV18E6). Each bar represents mean ± SEM (n = 6). Bars with different letters are significantly different from each other (P = 0.0461).


Effect of YAP mRNA levels in HT3 cells treated with or without E6 ( 400 nM ) for 60 min in McCoy ' s 5A with 1 % serum . Data were normalized with 18S mRNA . Bars with same letters are not significantly different from each other ( P > 0 . 05 ) . n = 4 , P = 0 . 9407 .

Effect of YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 60 min in McCoy's 5A with 1% serum. Data were normalized with 18S mRNA. Bars with same letters are not significantly different from each other (P > 0.05). n = 4, P = 0.9407.


YAP mRNA levels in HT3 cells treated with or without E6 ( 400 nM ) for 48 h in McCoy ' s 5A with 1 % serum . mRNA levels were measured with real - time PCR . Data were normalized with 18S mRNA . Each bar represents mean ± SEM ( n = 5 ) . Bars with same letters are not significantly different from each other ( P > 0 . 05 ) ( P = 0 . 3233 ) .

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YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 48 h in McCoy's 5A with 1% serum. mRNA levels were measured with real-time PCR. Data were normalized with 18S mRNA. Each bar represents mean ± SEM (n = 5). Bars with same letters are not significantly different from each other (P > 0.05) (P = 0.3233).


Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide ( CHX ) for 4 h or 8 h . β - actin was used as a protein loading control . Confluent HT3 cells were starved for 4 h before treatment with MG132 ( 10 μM ) or CHX ( 20 μg / ml ) for 0 , 4 or 8 h .

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Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.


HPV16E6 protein prevented YAP protein from degradation . Confluent HT3 cells were starved for 4 h before treatment with or without CHX ( 20 μg / ml ) , HPV16E6 , or CHX combined with HPV16E6 for 8 h . Treatment of starved HT3 cells with HPV16E6 ( 400 nM ) for 8 h suppressed degradation of YAP , but not EGFR protein . Quantitative data showing relative YAP protein levels in ( D ) . Protein levels were normalized with β - actin and presented as ratios relative to that of control . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( Ctrl vs . CHX , P = 0 . 0003 ; Ctrl vs . CHX + E6 , P = 0 . 2665 ; Ctrl vs . E6 , P = 0 . 0258 ) .

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HPV16E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16E6, or CHX combined with HPV16E6 for 8 h. Treatment of starved HT3 cells with HPV16E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).


Western blot analysis showing that expression of HPV16E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε , but decreased the protein level of SOCS6 . Importantly , HPV16E6 increased YAP phosphorylation at serine 127 , but suppressed its phosphorylation at serine 397 . HPV16E6 had no effect on the level of β - TrcP , LATS1 / 2 and MOB1 in HT3cervical cancer cells .

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Western blot analysis showing that expression of HPV16E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε, but decreased the protein level of SOCS6. Importantly, HPV16E6 increased YAP phosphorylation at serine 127, but suppressed its phosphorylation at serine 397. HPV16E6 had no effect on the level of β-TrcP, LATS1/2 and MOB1 in HT3cervical cancer cells.


Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18E6 siRNA ( siE6 ) reduced YAP protein levels , but increased SOCS6 protein levels .

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Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18E6 siRNA (siE6) reduced YAP protein levels, but increased SOCS6 protein levels.


Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA ( siSOCS # 1 and siSOCS6 # 2 ) increased total YAP protein , enhanced phosphorylation of YAP protein at serine 127 , but suppressed phosphorylation of YAP at serine 397 .

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Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA (siSOCS#1 and siSOCS6#2) increased total YAP protein, enhanced phosphorylation of YAP protein at serine 127, but suppressed phosphorylation of YAP at serine 397.


A - F Representative images showing expression of YAP ( in red ) in wild - type mousecervical tissues ( n = 5 ) ( A ) , HPV16E6 - induced ( C ) and HPV16E6 / E7 - induced ( E ) mousecervicaltumor tissue ( n = 4 each ) . Scale bars for ( A ) , ( C ) , and ( E ) : 100 μm . High - resolution images showing the expression and cellular location of YAP in normal cervical tissues ( B ) , HPV16E6 - induced ( D ) and HPV16E6 / E7 - induced ( F ) mousecervicaltumor tissue . Scale bars for ( B ) , ( D ) , and ( F ) : 25 μm .

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A-F Representative images showing expression of YAP (in red) in wild-type mousecervical tissues (n = 5) (A), HPV16E6-induced (C) and HPV16E6/E7-induced (E) mousecervicaltumor tissue (n = 4 each). Scale bars for (A), (C), and (E): 100 μm. High-resolution images showing the expression and cellular location of YAP in normal cervical tissues (B), HPV16E6-induced (D) and HPV16E6/E7-induced (F) mousecervicaltumor tissue. Scale bars for (B), (D), and (F): 25 μm.