M - P Representative of lectin stained choroidal flat mounts 7 14 days after coagulation in control mice ( M , N ) IFN - b treated ( O , P ) . Dashed lines indicate CNV areas asterisk marks central optic nerve head . Scale bar : 200 µmQ of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 7 - 12 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - : * * p = 0 . 0038 ) .

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

M-P Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in control mice (M, N) and IFN-b treated animals (O, P). Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µmQ Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 7-12 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: **p = 0.0038).


B - D Representative Iba1 results of retinal flat mounts detecting microglia / macrophages in spot 3 days after coagulation in Cx3cr1CreER ( B ) , Ifnar1fl / fl ( C ) Cx3cr1CreER : Ifnar1fl / fl ( D ) mice . Scale bar : 20 µm . E of amoeboid - shaped mononuclear phagocytes in spots . Values show mean ± SD . ( n = 17 - 29 retinas ; unpaired Student ' s t - : * * * p = 0 . 0003 , * * p = 0 . 0044 ) . F of immune in spots using a grid image system . Values show mean ± SD . ( n = 44 - 52 ; unpaired Student ' s t - : * * * p < 0 . 0001 ) .

O O O O B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Representative Iba1 staining results of retinal flat mounts detecting microglia/macrophages in laser spot 3 days after laser coagulation in Cx3cr1CreER (B), Ifnar1fl/fl (C) and Cx3cr1CreER:Ifnar1fl/fl (D) mice. Scale bar: 20 µm.E Quantification of amoeboid-shaped mononuclear phagocytes in laser spots. Values show mean ± SD. (n = 17-29 retinas; unpaired Student's t-test: ***p = 0.0003, **p = 0.0044).F Quantification of immune cell morphology in laser spots using a grid image analysis system. Values show mean ± SD. (n = 44-52 cells; unpaired Student's t-test: ***p < 0.0001).


G - O Representative fundus fluorescein angiography of Cx3cr1CreER ( G - I ) , Ifnar1fl / fl ( J - L ) Cx3cr1CreER : Ifnar1fl / fl ( M - O ) mice 3 , 7 14 days after - induced damage . P of vascular leakage by analyzing pixel intensities at 3 , 7 14 days after - induced retinal damage in Cx3cr1CreER , Ifnar1fl / fl Cx3cr1CreER : Ifnar1fl / fl mice . Values show mean ± SD . ( n = 5 - 12 eyes ; One - way ANOVA followed by Tukey ' s post - : * * p = 0 . 0032 , * p = 0 . 0247 ) .

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

G-O Representative fundus fluorescein angiography images of Cx3cr1CreER (G-I), Ifnar1fl/fl (J-L) and Cx3cr1CreER:Ifnar1fl/fl (M-O) mice 3, 7 and 14 days after laser-induced damage.P Quantification of vascular leakage by analyzing pixel intensities at 3, 7 and 14 days after laser-induced retinal damage in Cx3cr1CreER, Ifnar1fl/fl and Cx3cr1CreER:Ifnar1fl/fl mice. Values show mean ± SD. (n = 5-12 eyes; One-way ANOVA followed by Tukey's post-test: **p = 0.0032, *p = 0.0247).


Q - V Representative of lectin stained choroidal flat mounts 7 14 days after coagulation in Cx3cr1CreER ( Q , R ) , Ifnar1fl / fl ( S , T ) Cx3cr1CreER : Ifnar1fl / fl ( U , V ) mice . Dashed lines indicate CNV areas asterisk marks central optic nerve head . Scale bar : 200 µm . W of lectin - stained CNV areas with ImageJ software . Bars show mean ± SD . ( n = 4 - 11 RPE / choroidal flat mounts ; One - way ANOVA followed by Tukey ' s post - : * p = 0 . 043 , * * * p = 0 . 0007 ) .

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 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

Q-V Representative images of lectin stained choroidal flat mounts 7 and 14 days after laser coagulation in Cx3cr1CreER (Q, R), Ifnar1fl/fl (S, T) and Cx3cr1CreER:Ifnar1fl/fl (U, V) mice. Dashed lines indicate CNV areas and the asterisk marks the central optic nerve head. Scale bar: 200 µm.W Quantification of lectin-stained CNV areas with ImageJ software. Bars show mean ± SD. (n = 4-11 RPE/choroidal flat mounts; One-way ANOVA followed by Tukey's post-test: *p = 0.043, ***p = 0.0007).


( b ) of CTM - GFP mCTM - GFP relative to WT - GFP in HEK 24 h after transfections with WT - GFP , mCTM - GFP ( n = 6 ) or CTM - GFP . transfected with WT - GFP or CTM - GFP were treated without ( no treatment ; n = 13 ) or with serum deprivation ( + SD ; n = 5 ; to enhance CMA activity ) or with macroautophagy inhibitor 3 - methyladenine ( + 3 - MA ; 10 mM ; n = 5 ) , proteasome inhibitor ( + MG132 ; 5 μM ; n = 6 ) , lysosome inhibitor ( + NH4Cl ; 20 mM ; n = 6 ) or pepstatin A ( + Pep A ; 10 μM ; n = 5 ) . Top , representative immunoblots of lysates for GFP . Membranes reprobed for β - actin were used as a loading control . Bottom , means ± s . e . m . of relative , normalized to WT - GFP ( arbitrarily set as 1 ; bar ) ; n represents number of independent experiments from at least 4 separate cultures . One - way ANOVA with Fisher least significant difference , F ( 12 , 73 ) = 20 . 939 . * * * P 0 . 001 versus WT - GFP ( bar ) ; ΔΔΔP 0 . 001 relative to untreated CTM - GFP ( gray bar ) . Full - 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of CTM-GFP and mCTM-GFP relative to WT-GFP in HEK cells 24 h after individual transfections with WT-GFP, mCTM-GFP (n = 6) or CTM-GFP. Cells transfected with WT-GFP or CTM-GFP were treated without (no treatment; n = 13) or with serum deprivation (+ SD; n = 5; to enhance CMA activity) or with macroautophagy inhibitor 3-methyladenine (+ 3-MA; 10 mM; n = 5), proteasome inhibitor (+ MG132; 5 μM; n = 6), lysosome inhibitor (+ NH4Cl; 20 mM; n = 6) or pepstatin A (+ Pep A; 10 μM; n = 5). Top, representative immunoblots of cell lysates for GFP. Membranes reprobed for β-actin were used as a loading control. Bottom, means ± s.e.m. of relative protein levels, normalized to WT-GFP (arbitrarily set as 1; white bar); n represents the number of independent experiments from at least 4 separate cultures. One-way ANOVA with Fisher least significant difference test, F(12,73) = 20.939. ***P 0.001 versus WT-GFP (white bar); ΔΔΔP 0.001 relative to untreated CTM-GFP levels (gray bar). Full-length blots are presented in Supplementary Figure 9.


( c ) Representative confocal of colocalization of GFP with lysosome marker LAMP - 1 in COS - 7 transfected with either WT - GFP ( n = 17 ) or CTM - GFP ( n = 16 ) . Scale bar , 20 μm .

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

(c) Representative confocal images of colocalization of GFP with the lysosome marker LAMP-1 in COS-7 cells transfected with either WT-GFP (n = 17) or CTM-GFP (n = 16). Scale bar, 20 μm.


b ) Reciprocal coimmunoprecipitation ( IP ) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1 . Flag - tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA - GluN2Bct - CTM or HA - GluN2Bct - CTMm at various ratios in HEK , coimmunoprecipitation / or immunoblotting were performed 24 h thereafter . Anti - HA was used to detect HA - GluN2Bct - CTM HA - GluN2Bct - CTMm , anti - FLAG to detect wtDAPK1 cDAPK1 .

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

b) Reciprocal coimmunoprecipitation (IP) followed by immunoblotting revealed that GluN2Bct specifically interacted with cDAPK1 but not wtDAPK1. Flag-tagged inactive wtDAPK1 or constitutively active cDAPK1 was expressed with either HA-GluN2Bct-CTM or HA-GluN2Bct-CTMm at various ratios in HEK cells, and coimmunoprecipitation and/or immunoblotting were performed 24 h thereafter. Anti-HA was used to detect HA-GluN2Bct-CTM and HA-GluN2Bct-CTMm, anti-FLAG to detect wtDAPK1 and cDAPK1.


( c ) HA - GluN2Bct - CTM specifically dose - dependently decreased level of cDAPK1 ( n = 4 independent experiments from 4 separate cultures transfections ; P 0 . 001 ; F ( 5 , 18 ) = 18 . 27 ) but not

O O O O O B-MEASURED_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

(c) HA-GluN2Bct-CTM specifically and dose-dependently decreased the level of cDAPK1 (n = 4 independent experiments from 4 separate cell cultures and transfections; P 0.001; F(5,18) = 18.27) but not wtDAPK1


( d ; n = 3 independent experiments from 3 separate cultures transfections ; P = 0 . 933 ; F ( 5 , 12 ) = 0 . 249 ) . HA - GluN2Bct - CTM - mediated cDAPK1 knockdown was significantly reduced by NH4Cl ( c ; 20 mM ; n = 4 ; ΔΔΔP 0 . 001 compared to HA - GluN2Bct - CTM : cDAPK1 8 : 1 group ) by mutational inactivation of

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

(d; n = 3 independent experiments from 3 separate cell cultures and transfections; P = 0.933; F(5,12) = 0.249). HA-GluN2Bct-CTM-mediated cDAPK1 knockdown was significantly reduced by NH4Cl (c; 20 mM; n = 4; ΔΔΔP 0.001 compared to HA-GluN2Bct-CTM:cDAPK1 8:1 group) and by mutational inactivation of CTM


( e ; HA - GluN2Bct - CTMm ; P = 0 . 785 compared to control ( bar ) ; F ( 4 , 35 ) = 0 . 432 , 8 independent experiments from 8 separate cultures transfections ) . of cDAPK1 or wtDAPK1 cotransfected with pcDNA3 . 0 vector ( 0 : 1 , bar ) represent control values , arbitrarily set as 1 . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 , compared with control ; n . s . , not significant . Bars represent relative means ± s . e . m . Full - 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( pDAPK1 , n = 4 ) . Right , coimmunoprecipitation ( IP ) with anti - GluN2B sequential immunoblotting for DAPK1 GluN2B showed an NMDA - induced association between DAPK1 GluN2B ( n = 3 ) .

O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR 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 production of TAT - GluN2Bct - CTM TAT - GluN2Bct peptides ( left ) using an E . coli expression system . Coomassie blue after SDS - PAGE assessed their purity ( right ) ; left lane , marker .

O O O O O O O O B-MEASURED_VAR 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

(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 , this was prevented by NH4Cl ( 20 mM ; n = 5 ; one - way ANOVA ; P 0 . 001 , F ( 5 , 36 ) = 10 . 891 ) dose - ( d , doses in μM ; n = 4 ; P 0 . 001 ; F ( 6 , 21 ) = 18 . 14 ) 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 during 30 - min NMDA stimulation ) produced a transient reduction of DAPK1 , returning to baseline within 7 h ( n = 4 ; P = 0 . 888 ) , an additional dose of 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O 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 B-CONTROLLED_VAR 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

(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 TAT - GluN2BCTM .

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

(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 , this was prevented by NH4Cl ( 20 mM ; n = 5 ; P = 0 . 302 ) . One - way ANOVA , P 0 . 001 , F ( 5 , 24 ) = 13 . 591 . Relative of DAPK1 were normalized to those in saline control group compared to saline control ( bar ; * P 0 . 05 , * * P 0 . 01 * * * P 0 . 001 ) or NMDA - treated group ( gray bar ; ΔP 0 . 05 , ΔΔP 0 . 01 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 experiments . Full - 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 B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

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

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

(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 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 α - synuclein ( bottom ) . NH4Cl rescued PSD - 95 degradation . Sample sizes represent experiments from at least 2 separate cultures . reprobing for β - actin was used as an additional specificity loading control . ΔP 0 . 05 , * * , ΔΔP 0 . 01 * * * P 0 . 001 ; bars represent relative mean values ± s . e . m . normalized to saline control ( arbitrarily set as 1 ) . Full - blots are presented in Supplementary Figure 9 .

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

(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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 during H2O2 treatment ( 300 μM ; 30 min ) knocked down DAPK1 at 2 h after washout , 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 relative to saline control group . β - actin was used as a loading control .

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 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 B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_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 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 B-MEASURED_VAR 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 revealed that H2O2 treatment ( 300 μM ; 30 min ) resulted in a significant increase in neuronal 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 maintained throughout 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 NMDAR antagonist APV ( 1 μM ; n = 4 ; 2 . 16 ± 0 . 14 ; P = 0 . 169 to H2O2 group ) . NH4Cl abolished 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 saline control ( bar , arbitrarily set as 1 ) . n represents experiments from at least 3 separate cultures . Full - blots are presented in Supplementary Figure 9 .

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

(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 of a series of transverse brain sections showed reliable damage in 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

(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 infarct ( Ipsi ) but not ( 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 - Student ' s t - * * * P 0 . 001 ; n . s . , not significant .

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 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

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

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 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 B-MEASURED_VAR 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 , of damage by counting number of Fluorojade B - positive in each 640 × 640 pixel image at 10 × magnification . TAT - GluN2BCTM ( 10 mg per kilogram ) displayed more prominent neuroprotection in cortex ( P 0 . 001 ) 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 collected from at least 2 litters . Scale bars 1 mm in b , c , e , 20 μm in f . Full - 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

(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.


( L ) Larval NSCs of control ( ctrl ; yw ) , pr - set7716 / Df , UAS - pr - set7 induced with insc - Gal4 at 72h ALH were stained for Pr - set7 , Mira . ( M , N ) Quantifications of NSC diameters ( M ) at 24h ALH EdU incorporation ( N ) for genotypes in ( O ) . In ( M ) , n = 152 for control ; n = 129 for pr - set7716 / Df . In ( N ) , for control , n = 601 , 13 BL at 24h ALH ; n = 988 , 14 BL at 48h ALH ; n = 813 , 9 BL at 72h ALH n = 829 , 10 BL at 96h ALH . For pr - set7716 / Df , n = 566 , 12 BL at 24h ALH ; n = 808 , 13 BL at 48h ALH ; n = 1032 , 10 BL at 72h ALH n = 825 , 9 BL at 96h ALH . Data information : data are presented in as mean ± SD M , N ) . n numbers in N ) were total NSCs counted with total brain lobes measured , while each brain lobe considered as one biological replicate . arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann - Whitney M , N ) . * * * P < 0 . 001 . Scale bars , 5 μm L ) ,

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

(L) Larval NSCs of control (ctrl; yw), pr-set7716/Df, and UAS-pr-set7 induced with insc-Gal4 at 72h ALH were stained for Pr-set7, Mira and DNA. (M, N) Quantifications of NSC diameters (M) at 24h ALH and EdU incorporation (N) for genotypes in (O). In (M), n=152 for control; n=129 for pr-set7716/Df. In (N), for control, n=601, 13 BL at 24h ALH; n=988, 14 BL at 48h ALH; n=813, 9 BL at 72h ALH and n=829, 10 BL at 96h ALH. For pr-set7716/Df, n=566, 12 BL at 24h ALH; n=808, 13 BL at 48h ALH; n=1032, 10 BL at 72h ALH and n=825, 9 BL at 96h ALH. Data information: Quantification data are presented in as mean ± SD M, N). n numbers in N) were total NSCs counted with total brain lobes measured, while each brain lobe considered as one biological replicate. White arrows point to NSCs Statistical analyses were done comparing two different genotypes using Mann-Whitney test M, N). ***P < 0.001. Scale bars, 5 μm L),


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

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

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


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

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


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

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

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


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

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-MEASURED_VAR O B-MEASURED_VAR I-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

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


( H , I ) Quantifications of Ebd1 ( H ) corrected total ( CCF ) ( I ) In ( H ) , n = 756 , 9 BL for control ; n = 765 , 9 BL for pr - set7716 / Df . In ( I ) , CCF = Integrated Density - ( of selected × Mean of background readings ) . CCFs were 1102 . 2 ± 493 . 2 ( n = 99 ) for control ( ctrl ; yw ) 144 . 0 ± 88 . 0 ( n = 98 ) for 716 / Df . Data information : Data are presented in , I , s mean ± SD . n numbers in ( H ) were total NSCs counted with total brain lobes measured , while each brain lobe was considered as one biological replicate . Statistical analyses were done comparing two different genotypes using Mann - Whitney , H , I , * * * P < 0 . 001 .

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

(H, I) Quantifications of nuclear Ebd1 (H) and the corrected total cell fluorescence (CCF) (I) In (H), n=756, 9 BL for control; n=765, 9 BL for pr-set7716/Df. In (I), CCF = Integrated Density - (Area of selected cell × Mean fluorescence of background readings). CCFs were 1102.2±493.2 (n=99) for control (ctrl; yw) and 144.0±88.0 (n=98) for 716/Df. Data information: Data are presented in , I, s mean ± SD. n numbers in (H) were total NSCs counted with total brain lobes measured, while each brain lobe was considered as one biological replicate. Statistical analyses were done comparing two different genotypes using Mann-Whitney test , H, I, ***P < 0.001.


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O B-MEASURED_VAR 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 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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O B-CONTROLLED_VAR 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

O O O O O O 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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


FRhK4 infected with SIV - VSV - luc analyzed by

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


Rat2 infected with MuLV - VSV - luc analyzed by

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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