Figure 5A , B ) Mean baseline - detrended bioluminescence profiles from plate wells containing either PER2 : : LUC U2OS cells with ( WT , left panel ) or without REV - ERBα ( KO , right panel ) recorded at 37 ° C before and after transfer to 18 ° C for 24h ( A ) or 5h ( B ) ( grey region ) from biological repeat 1 . 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18 ° C exposure ( time zero ) . D ) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37 ° C or transferred to 18 ° C for 24h ( top panel ) or 5h ( bottom panel ) from biological repeats 1 and 2 . Points are colored according to their distinct phase prior to 18 ° C exposure . Colored lines ( bottom panel ) show the change in the mean for the points from each phase . Boxplots display median ( central line ) , interquartile range ( IQR , box edges ) and range ( whiskers ) . Number of wells : top panel WT cells : 37 ° C ( 120 ) , 18 ° C 24h ( 119 ) ; top panel KO cells : 37 ° C ( 120 ) , 18 ° C 24h ( 120 ) ; bottom panel WT cells : 37 ° C ( 58 ) , 18 ° C 5h ( 120 ) ; top panel KO cells : 37 ° C ( 60 ) , 18 ° C 5h ( 120 ) . E ) Bar graphs showing the mean amplitude change ( left axis ) following 18 ° C exposure for 5h for each group of plate well profiles ( grouped according to their distinct phase ( a - f ) prior to 18 ° C exposure , 10 wells per group ) for both WT and KO cells from biological repeats 1 and 2 . Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2 : LUC ( dashed line ) and predicted REV - ERBα expression level ( right axis ) ( grey shaded region ( light grey indicates REV - ERBα deletion ) ) .

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

Figure 5A,B) Mean baseline-detrended bioluminescence profiles from plate wells containing either PER2::LUC U2OS cells with (WT, left panel) or without REV-ERBα (KO, right panel) recorded at 37°C before and after transfer to 18°C for 24h (A) or 5h (B) (grey region) from biological repeat 1. 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18°C exposure (time zero).D) Boxplots and individual points for times for each plate well profile for both WT and KO cells kept at 37°C or transferred to 18°C for 24h (top panel) or 5h (bottom panel) from biological repeats 1 and 2. Points are colored according to their distinct phase prior to 18°C exposure. Colored lines (bottom panel) show the change in the mean for the points from each phase. Boxplots display median (central line), interquartile range (IQR, box edges) and range (whiskers). Number of wells: top panel WT cells: 37°C (120), 18°C 24h (119); top panel KO cells: 37°C (120), 18°C 24h (120); bottom panel WT cells: 37°C (58), 18°C 5h (120); top panel KO cells: 37°C (60), 18°C 5h (120).E) Bar graphs showing the mean amplitude change (left axis) following 18°C exposure for 5h for each group of plate well profiles (grouped according to their distinct phase (a-f) prior to 18°C exposure, 10 wells per group) for both WT and KO cells from biological repeats 1 and 2. Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2:LUC (dashed line) and predicted REV-ERBα expression level (right axis) (grey shaded region (light grey indicates REV-ERBα deletion)).


Figure 1 ( A ) Upper panel : immunoblot analysis of COS7 cells treated for 12 h with MG132 ( 25 μM ) and vinblastine ( 25 μM ) . Lower panel : real ‐ time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure ( 25 μM , 12 h ) . ( B , C ) Immunofluorescence staining of indicated proteins in COS7 cells treated as in ( A ) . Scale bar , 20 μm . ( D ) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 ( 25 μM , 12 h ) . Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online ) . Diagram shows the percentage of aggresome ‐ positive cells . ( E ) Aggresome quantification as in supplementary Fig S1D online from cells analysed in ( H ) . Images are shown in supplementary Fig S1G online . Diagram shows the percentage of aggresome ‐ positive cells at 4 , 24 and 48 h after MG132 wash out . ( F ) Cells were treated as in ( H ) ; however , 4 h after MG132 wash out , BafA1 ( 5 μM ) was added for 20 h to inhibit aggresome clearance . Diagrams show the percentage of aggresome ‐ positive cells and the mean size of aggresomes ( supplementary Fig S1H online ) . Images are shown in supplementary Fig S1G online . ( G ) Dot ‐ blot ( upper panel , quantification in supplementary Fig S1B online ) and western blot ( lower panel ) analyses of 0 . 5 % NP40 ‐ soluble and ‐ insoluble proteins from cells shown in Fig 1D ; supplementary Fig S1D online . ( H ) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 ( 25 μM , 18 h ) or DMSO as a control . After MG132 wash out , cells were allowed to recover for 4 , 24 and 48 h and 0 . 5 % NP40 ‐ soluble and ‐ insoluble proteins were analysed by immunoblotting . ( I ) COS7 cells were transfected for 24 h with GST or GST fused to DIC ( GST ‐ DIC ) and treated with MG132 ( 25 μM , 12 h ) . Indicated proteins in whole ‐ cell lysates and in precipitates obtained by GST pull ‐ down with glutathione beads were analysed by immunoblotting . BafA1 , bafilomycin A1 ; ctrl , control ; DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; Hsp70 , heat ‐ shock protein 70 ; nons , nonsense ; siRNA , short ‐ interfering RNA ; vinblast , vinblastine .

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

Figure 1(A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real‐time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h).(B,C) Immunofluorescence staining of indicated proteins in COS7 cells treated as in (A). Scale bar, 20 μm.(D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitinimmunostaining ( supplementary Fig S1D online). Diagram shows the percentage of aggresome‐positive cells.(E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome‐positive cells at 4, 24 and 48 h after MG132 wash out.(F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome‐positive cells and the mean size of aggresomes ( supplementary Fig S1H online). Images are shown in supplementary Fig S1G online.(G) Dot‐blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40‐soluble and ‐insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online.(H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40‐soluble and ‐insoluble proteins were analysed by immunoblotting.(I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST‐DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole‐cell lysates and in precipitates obtained by GST pull‐down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; Hsp70, heat‐shock protein 70; nons, nonsense; siRNA, short‐interfering RNA; vinblast, vinblastine.


Figure 2 ( A ) GST pull ‐ down analysis as in Fig 1I , but performed in cells transfected for 36 h with full ‐ length FLAG ‐ BAG3 ( I ) or the BAG3 deletion mutants FLAG - ( Δ421 - 498 ) - BAG3 ( II ) , FLAG - BAG ‐ domain ( III ) , and FLAG - PxxP - BAG ‐ domain ( IV ) . Asterisk , nonspecific band . ( B ) HEK cells were transfected for 18 h with SODG85R ‐ GFP , along with the indicated FLAG ‐ tagged BAG3 constructs ( shown in A ) and HA ‐ tagged Hsp70 ( HA ‐ Hsp70 ) . SODG85R ‐ GFP showed three distinct localization patterns : cytoplasmic , pre ‐ aggresomal and aggresomal ( see representative pictures on the right and supplementary Fig S2F online ) . The percentages of cells bearing pre ‐ aggresomal ( upper panel ) and aggresomal ( lower panel ) SODG85R ‐ GFP are shown . Scale bar , 10 μm . ( C ) HEK cells transfected with SODG85R ‐ GFP , BAG3 and Hsp70 were additionally transfected with FLAG ‐ p50 , as indicated . Diagrams show the percentages of cells with pre ‐ aggresomal and aggresomal SODG85R ‐ GFP . Right panel : representative image showing pre ‐ aggresomal localization of FLAG ‐ p50 and SODG85R ‐ GFP . Scale bar , 5 μm . ( D ) GST pull ‐ down analysis as in ( A ) , but in HEK cells transfected for 18 h with SODWT ‐ GFP or mutant SODG85R ‐ GFP , along with FLAG ‐ BAG3 . ( E ) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA . At 6 h after transfection , lactacystin ( 10 μM ) or GST as vehicle control was added for GST and GST pull ‐ down analysis was performed as in ( D ) . ( F ) Upper panel : sucrose ‐ density gradient analysis of extracts from HEK cells transfected with SODG85R ‐ GFP and GST ‐ DIC , along with FLAG ‐ BAG3 or vector control . Note the immunoreactive bands in high ‐ density fraction 14 on BAG3 overexpression ( arrow ) . Migration of a 20S marker protein is indicated . Lower panel : GST ‐ DIC pull ‐ down analyses of dynein complexes present in sucrose fraction 6 and 14 . ( G ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells transfected with FLAG ‐ tagged BAG3 constructs ( I - IV ; indicated as in A ) . ( H ) GST pull ‐ down analysis of SODG85R ‐ GFP as in ( D ) but in cells treated with PYR41 ( 10 μM ; 12 h ) , as indicated . DIC , dynein intermediate chain ; DMSO , dimethyl sulphoxide ; GST , glutathione ‐ S ‐ transferase ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short ‐ interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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

Figure 2(A) GST pull‐down analysis as in Fig 1I, but performed in cells transfected for 36 h with full‐length FLAG‐BAG3 (I) or the BAG3 deletion mutants FLAG-(Δ421-498)-BAG3 (II), FLAG-BAG‐domain (III), and FLAG-PxxP-BAG‐domain (IV). Asterisk, nonspecific band.(B) HEK cells were transfected for 18 h with SODG85R‐GFP, along with the indicated FLAG‐tagged BAG3 constructs (shown in A) and HA‐tagged Hsp70 (HA‐Hsp70). SODG85R‐GFP showed three distinct localization patterns: cytoplasmic, pre‐aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre‐aggresomal (upper panel) and aggresomal (lower panel) SODG85R‐GFP are shown. Scale bar, 10 μm.(C) HEK cells transfected with SODG85R‐GFP, BAG3 and Hsp70 were additionally transfected with FLAG‐p50, as indicated. Diagrams show the percentages of cells with pre‐aggresomal and aggresomal SODG85R‐GFP. Right panel: representative image showing pre‐aggresomal localization of FLAG‐p50 and SODG85R‐GFP. Scale bar, 5 μm.(D) GST pull‐down analysis as in (A), but in HEK cells transfected for 18 h with SODWT‐GFP or mutant SODG85R‐GFP, along with FLAG‐BAG3.(E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or GST as vehicle control was added for GST and GST pull‐down analysis was performed as in (D).(F) Upper panel: sucrose‐density gradient analysis of extracts from HEK cells transfected with SODG85R‐GFP and GST‐DIC, along with FLAG‐BAG3 or vector control. Note the immunoreactive bands in high‐density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST‐DIC pull‐down analyses of dynein complexes present in sucrose fraction 6 and 14.(G) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells transfected with FLAG‐tagged BAG3 constructs (I-IV; indicated as in A).(H) GST pull‐down analysis of SODG85R‐GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione‐S‐transferase; HA, haemagglutinin; Hsp70, heat‐shock protein 70; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short‐interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


Figure 3 ( A ) Analysis of SODG85R ‐ GFP ( green ) localization with aggresome marker vimentin , the autophagic receptors p62 and NBR1 , the autophagosome marker LC3 and the lysosomal marker LAMP2 ( all in red ) in BAG3 / Hsp70 ‐ transfected HEK cells . Representative merged images of cells with cytoplasmic , pre ‐ aggresomal and aggresomal SOD distribution are shown . Arrows indicate LC3 ‐ positive autophagosomes . Scale bar , 5 μm . ( B ) Transmission electron microscope analysis of SODG85R ‐ GFP aggresome in cells transfected as in ( A ) . Marked area in ( a ) is shown magnified in ( b ) . Arrows , autophagosomes . Arrowheads , autolysosomes . Scale bar , 1 μm . ( C ) HEK cells transfected for 8 h with SODG85R ‐ GFP along with FLAG ‐ BAG3 and HA ‐ Hsp70 , as indicated , were treated for 24 h with BafA1 ( 0 . 1 μM ) . Indicated proteins were analysed by immunoblotting . Diagram shows the ratio of SODG85R ‐ GFP levels in BafA1 ‐ treated cells to those in untreated cells . ( D ) Same analysis as in ( C ) but with SODWT ‐ GFP . ( E ) Similar analysis as in ( C ) but in cells transfected 24 h before with nonsense or atg7 siRNA . ( F ) HEK cells were transfected with SODG85R ‐ GFP and indicated FLAG ‐ tagged BAG3 constructs together with HA ‐ Hsp70 , and lysosomal degradation of SODG85R ‐ GFP was examined as in ( C ) . Corresponding immunoblots are shown in supplementary Fig S3F online . Ag , aggresome ; BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; HA , haemagglutinin ; Hsp70 , heat ‐ shock protein 70 ; N , nucleus ; NS , not significant ; nons , nonsense ; PxxP , proline ‐ rich repeat ; siRNA , short interfering RNA ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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

Figure 3(A) Analysis of SODG85R‐GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70‐transfected HEK cells. Representative merged images of cells with cytoplasmic, pre‐aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3‐positive autophagosomes. Scale bar, 5 μm.(B) Transmission electron microscope analysis of SODG85R‐GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm.(C) HEK cells transfected for 8 h with SODG85R‐GFP along with FLAG‐BAG3 and HA‐Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R‐GFP levels in BafA1‐treated cells to those in untreated cells.(D) Same analysis as in (C) but with SODWT‐GFP.(E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA.(F) HEK cells were transfected with SODG85R‐GFP and indicated FLAG‐tagged BAG3 constructs together with HA‐Hsp70, and lysosomal degradation of SODG85R‐GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat‐shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline‐rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild‐type SOD.


Figure 4 ( A ) Spinal cord sections of mice overexpressing SODWT and SODG85R ( disease end ‐ stage ) were immunostained for BAG3 and SOD1 . Arrows indicate SOD1 ‐ and BAG3 ‐ positive aggregates in the neuropil . Scale bar , 20 μm . ( B ) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset . Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R . Scale bar , 10 μm . Magnific . , magnification ; SOD , superoxide dismutase ; SODWT , wild ‐ type SOD .

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

Figure 4(A) Spinal cord sections of mice overexpressing SODWT and SODG85R (disease end‐stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1‐ and BAG3‐positive aggregates in the neuropil. Scale bar, 20 μm.(B) Localization analysis of BAG3 and SODG85R in surviving spinal cord motor neurons fromSODG85Rmice at the time of disease symptom onset. Arrows indicate perinuclear aggresomes containing BAG3 and SODG85R. Scale bar, 10 μm. Magnific., magnification; SOD, superoxide dismutase; SODWT, wild‐type SOD.


figf1 ( 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 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 .

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

figf1(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 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.


figf2b ) 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 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 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 ( 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 .

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

figf2b) 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 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 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 (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.


figf3 ( a ) Left , NMDA ( 50 μM ; 30 min ) activated DAPK1 , resulting in a time - dependent decrease in its phosphorylation levels ( pDAPK1 , n = 4 ) . Right , coimmunoprecipitation ( IP ) with anti - GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA - induced association between DAPK1 and GluN2B ( n = 3 ) . ( b ) Design and production of TAT - GluN2Bct - CTM and TAT - GluN2Bct peptides ( left ) using an E . coli expression system . Coomassie blue staining after SDS - PAGE assessed their purity ( right ) ; left lane , size marker . ( c ) Bath application of TAT - GluN2Bct - CTM ( 200 μM ; n = 9 ) , but not TAT - GluN2Bct ( 200 μM ; n = 6 ) , knocked down activated DAPK1 , and this was prevented by NH4Cl ( 20 mM ; n = 5 ; one - way ANOVA ; P 0 . 001 , F ( 5 , 36 ) = 10 . 891 ) and dose - ( d , doses in μM ; n = 4 ; P 0 . 001 ; F ( 6 , 21 ) = 18 . 14 ) and time - dependent ( e ; P 0 . 001 ; F ( 8 , 44 ) = 12 . 074 ) . ( f ) A single pretreatment of TAT - GluN2Bct - CTM ( sing ; 200 μM , 60 min before and during the 30 - min NMDA stimulation ) produced a transient reduction of DAPK1 , returning to baseline within 7 h ( n = 4 ; P = 0 . 888 ) , and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h ( mult ; n = 4 ; ΔΔP = 0 . 002 ) . One - way ANOVA ; P 0 . 001 , F ( 4 , 15 ) = 10 . 389 . ( g ) Synthetic peptides TAT - GluN2B and TAT - GluN2BCTM . ( h ) TAT - GluN2BCTM ( 25 μM ; n = 5 ; P = 0 . 001 ) , but not control TAT - GluN2B ( 25 μM ; n = 4 ; P = 0 . 223 ) , decreased native DAPK1 , and this was prevented by NH4Cl ( 20 mM ; n = 5 ; P = 0 . 302 ) . One - way ANOVA , P 0 . 001 , F ( 5 , 24 ) = 13 . 591 . Relative levels of DAPK1 were normalized to those in the saline control group and compared to the saline control ( white bar ; * P 0 . 05 , * * P 0 . 01 and * * * P 0 . 001 ) or NMDA - treated group ( gray bar ; ΔP 0 . 05 , ΔΔP 0 . 01 and ΔΔΔP 0 . 001 ) . Membranes reprobed for β - actin were used as a loading control . Bars represent relative means ± s . e . m . Sample sizes represent number of individual experiments . Full - length blots are presented in Supplementary Figure 9 .

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

figf3(a) Left, NMDA (50 μM; 30 min) activated DAPK1, resulting in a time-dependent decrease in its phosphorylation levels (pDAPK1, n = 4). Right, coimmunoprecipitation (IP) with anti-GluN2B and sequential immunoblotting for DAPK1 and GluN2B showed an NMDA-induced association between DAPK1 and GluN2B (n = 3).(b) Design and production of TAT-GluN2Bct-CTM and TAT-GluN2Bct peptides (left) using an E. coli expression system. Coomassie blue staining after SDS-PAGE assessed their purity (right); left lane, size marker.(c) Bath application of TAT-GluN2Bct-CTM (200 μM; n = 9), but not TAT-GluN2Bct (200 μM; n = 6), knocked down activated DAPK1, and this was prevented by NH4Cl (20 mM; n = 5; one-way ANOVA; P 0.001, F(5,36) = 10.891) and dose- (d, doses in μM; n = 4; P 0.001; F(6,21) = 18.14) and time-dependent (e; P 0.001; F(8,44) = 12.074). (f) A single pretreatment of TAT-GluN2Bct-CTM (sing; 200 μM, 60 min before and during the 30-min NMDA stimulation) produced a transient reduction of DAPK1, returning to baseline within 7 h (n = 4; P = 0.888), and an additional dose of the peptide after NMDA washout resulted in a persistent decrease in DAPK1 up to 7 h (mult; n = 4; ΔΔP = 0.002). One-way ANOVA; P 0.001, F(4,15) = 10.389.(g) Synthetic peptides TAT-GluN2B and TAT-GluN2BCTM.(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.


figf4 ( a ) Top , the synthetic cell - penetrating α - synuclein targeting peptide TAT - βsynCTM and its control TAT - βsyn . Middle , immunoblots demonstrate that TAT - βsynCTM ( 25 μM ; n = 5 ) , but not the CTM - lacking control peptide TAT - βsyn ( 25 μM ; n = 5 ) , specifically decreased the targeted endogenous α - synuclein ( one - way ANOVA , Tukey post hoc , P 0 . 001 , F ( 3 , 16 ) = 12 . 435 ) , without affecting the level of unrelated control protein PSD - 95 at 4 h ( bottom ) , and this reduction was prevented in the presence of NH4Cl ( 20 mM ; n = 5 ) . Sample sizes represent individual experiments from at least 3 separate primary cultures . ( ( b ) Top , PSD - 95 targeting peptide TAT - GluN2B9cCTM and control TAT - GluN2B9c . Middle , TAT - GluN2B9cCTM ( 25 μM ; n = 4 ) , but not Tat - GluN2B9c ( 25 μM ; n = 4 ) , effectively degraded endogenous PSD - 95 ( one - way ANOVA , Tukey post hoc , P 0 . 001 , F ( 3 , 12 ) = 18 . 154 ) without perturbing untargeted protein α - synuclein ( bottom ) . NH4Cl rescued PSD - 95 degradation . Sample sizes represent individual experiments from at least 2 separate primary cultures . Membrane reprobing for β - actin was used as an additional specificity and loading control . ΔP 0 . 05 , * * , ΔΔP 0 . 01 and * * * P 0 . 001 ; bars represent relative mean values ± s . e . m . normalized to the saline control ( arbitrarily set as 1 ) . Full - length blots are presented in Supplementary Figure 9 .

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

figf4(a) Top, the synthetic cell-penetrating α-synuclein targeting peptide TAT-βsynCTM and its control TAT-βsyn. Middle, immunoblots demonstrate that TAT-βsynCTM (25 μM; n = 5), but not the CTM-lacking control peptide TAT-βsyn (25 μM; n = 5), specifically decreased the targeted endogenous α-synuclein (one-way ANOVA, Tukey post hoc, P 0.001, F(3,16) = 12.435), without affecting the level of unrelated control protein PSD-95 at 4 h (bottom), and this reduction was prevented in the presence of NH4Cl (20 mM; n = 5). Sample sizes represent individual experiments from at least 3 separate primary cultures. ((b) Top, PSD-95 targeting peptide TAT-GluN2B9cCTM and control TAT-GluN2B9c. Middle, TAT-GluN2B9cCTM (25 μM; n = 4), but not Tat-GluN2B9c (25 μM; n = 4), effectively degraded endogenous PSD-95 (one-way ANOVA, Tukey post hoc, P 0.001, F(3,12) = 18.154) without perturbing untargeted protein α-synuclein (bottom). NH4Cl rescued PSD-95 degradation. Sample sizes represent individual experiments from at least 2 separate primary cultures. Membrane reprobing for β-actin was used as an additional specificity and loading control. ΔP 0.05, **,ΔΔP 0.01 and ***P 0.001; bars represent relative mean values ± s.e.m. normalized to the saline control (arbitrarily set as 1). Full-length blots are presented in Supplementary Figure 9.


figf5 ( a ) Immunoblotting for phosphorylated DAPK1 ( pDAPK1 ) revealed a time - dependent activation ( dephosphorylation ) of DAPK1 by H2O2 treatment ( 300 μM ; 30 min ; n = 4 ) . ( b ) Bath application of 100 μM TAT - GluN2Bct - CTM ( 36 . 54 ± 7 . 1 % of control ; n = 8 ; P = 0 . 001 ) , but not TAT - GluN2Bct ( 89 . 13 ± 10 . 78 % ; n = 7 ; P = 0 . 311 ) , 60 min before and during H2O2 treatment ( 300 μM ; 30 min ) knocked down DAPK1 at 2 h after washout , and this effect was rescued by NH4Cl ( 20 mM ; n = 8 ; * * P = 0 . 003 to control ; P = 0 . 106 to H2O2 - treated ) . One - way ANOVA , F ( 4 , 34 ) = 11 . 628 , P 0 . 001 . Bars represent DAPK1 levels relative to saline control group . β - actin was used as a loading control . ( c ) LDH assay revealed that H2O2 treatment ( 300 μM ; 30 min ) resulted in a significant increase in neuronal death 12 h after treatment ( n = 8 ; 2 . 50 ± 0 . 12 ; P 0 . 001 to control ) , which was rescued by breaking down H2O2 with catalase ( 100 U in 10 μl phosphate - buffered saline ; n = 4 ; 1 . 17 ± 0 . 02 ; P = 0 . 001 to H2O2 group ) . H2O2 - induced neurotoxicity was significantly reduced by TAT - GluN2Bct - CTM ( 50 μM ; applied 60 min before and maintained throughout the experiments ; n = 9 ; 1 . 56 ± 0 . 08 ; P = 0 . 001 to H2O2 group ) , but not by TAT - GluN2Bct ( 50 μM ; n = 9 ; 2 . 63 ± 0 . 10 ; P = 0 . 105 to H2O2 group ) or the NMDAR antagonist APV ( 1 μM ; n = 4 ; 2 . 16 ± 0 . 14 ; P = 0 . 169 to H2O2 group ) . NH4Cl abolished the neuroprotective effect of TAT - GluN2Bct - CTM ( n = 4 ; 2 . 39 ± 0 . 27 ; P = 0 . 538 compared to H2O2 group ) . One - way ANOVA , P 0 . 001 , F ( 6 , 41 ) = 26 . 842 . * * * , ΔΔΔP 0 . 001 ; bars represent relative mean values ± s . e . m . normalized to the saline control ( white bar , arbitrarily set as 1 ) . n represents individual experiments from at least 3 separate primary cultures . Full - length blots are presented in Supplementary Figure 9 .

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

figf5(a) Immunoblotting for phosphorylated DAPK1 (pDAPK1) revealed a time-dependent activation (dephosphorylation) of DAPK1 by H2O2 treatment (300 μM; 30 min; n = 4).(b) Bath application of 100 μM TAT-GluN2Bct-CTM (36.54 ± 7.1% of control; n = 8; P = 0.001), but not TAT-GluN2Bct (89.13 ± 10.78%; n = 7; P = 0.311), 60 min before and during H2O2 treatment (300 μM; 30 min) knocked down DAPK1 at 2 h after washout, and this effect was rescued by NH4Cl (20 mM; n = 8; **P = 0.003 to control; P = 0.106 to H2O2-treated). One-way ANOVA, F(4,34) = 11.628, P 0.001. Bars represent DAPK1 levels relative to saline control group. β-actin was used as a loading control.(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.


figf6 ( b ) 2 , 3 , 5 - Triphenyltetrazolium chloride staining of a series of transverse brain sections showed reliable damage in the ipsilateral side following unilateral MCAo . ( c ) Black dashed lines on a section stained as in b represent brain areas removed for immunoblotting . ( d ) Immunoblots demonstrated specific DAPK1 knockdown in the infarct ( Ipsi ) but not contralateral ( Contra ) side following application of TAT - GluN2BCTM ( 10 mg per kilogram , i . v . ; n = 3 ; t ( 4 ) = 14 . 459 , P 0 . 001 ) but not TAT - GluN2B ( 10 mg per kilogram , i . v . ; n = 3 ; t ( 4 ) = 0 . 739 , P = 0 . 501 ) . β - actin was used as a loading control ; two - tailed Student ' s t - test * * * P 0 . 001 ; n . s . , not significant . ( e ) HE ( left ) and immunohistochemical DAPK1 ( right ) staining of adjacent brain sections . Compared with those in saline ( top ) and TAT - GluN2B - treated ( middle ) controls , TAT - GluN2BCTM treatment ( bottom ) selectively reduced infarct area ( left ) and DAPK1 levels ( right ) ipsilaterally . Black outlining delineates infarct areas as visualized with HE staining . ( f ) Left , brain sections stained with Fluorojade B in rats injected with saline ( n = 6 ) , TAT - GluN2B ( n = 5 ) or TAT - GluN2BCTM ( n = 5 ) after treatment as shown in a . Right , quantification of cellular damage by counting the number of Fluorojade B - positive cells in each 640 × 640 pixel image at 10 × magnification . TAT - GluN2BCTM ( 10 mg per kilogram ) displayed more prominent neuroprotection in the cortex ( P 0 . 001 ) and striatum ( P 0 . 001 ) as compared to TAT - GluN2B ( 10 mg per kilogram ) . Cortex : H ( 2 ) = 41 . 235 ; P 0 . 001 ; striatum : H ( 2 ) = 38 . 808 ; P 0 . 001 . Kruskal - Wallis ANOVA on ranks with Dunn ' s post hoc ; bars represent relative means ± s . e . m . , * * * , ΔΔΔP 0 . 001 . n values represent tissue from 3 animals collected from at least 2 litters . Scale bars 1 mm in b , c , e , 20 μm in f . Full - length blots are presented in Supplementary Figure 9 .

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

figf6(b) 2,3,5-Triphenyltetrazolium chloride staining of a series of transverse brain sections showed reliable damage in the ipsilateral side following unilateral MCAo. (c) Black dashed lines on a section stained as in b represent brain areas removed for immunoblotting.(d) Immunoblots demonstrated specific DAPK1 knockdown in the infarct (Ipsi) but not contralateral (Contra) side following application of TAT-GluN2BCTM (10 mg per kilogram, i.v.; n = 3; t(4) = 14.459, P 0.001) but not TAT-GluN2B (10 mg per kilogram, i.v.; n = 3; t(4) = 0.739, P = 0.501). β-actin was used as a loading control; two-tailed Student's t-test ***P 0.001; n.s., not significant.(e) HE (left) and immunohistochemical DAPK1 (right) staining of adjacent brain sections. Compared with those in saline (top) and TAT-GluN2B-treated (middle) controls, TAT-GluN2BCTM treatment (bottom) selectively reduced infarct area (left) and DAPK1 levels (right) ipsilaterally. Black outlining delineates infarct areas as visualized with HE staining.(f) Left, brain sections stained with Fluorojade B in rats injected with saline (n = 6), TAT-GluN2B (n = 5) or TAT-GluN2BCTM (n = 5) after treatment as shown in a. Right, quantification of cellular damage by counting the number of Fluorojade B-positive cells in each 640 × 640 pixel image at 10× magnification. TAT-GluN2BCTM (10 mg per kilogram) displayed more prominent neuroprotection in the cortex (P 0.001) and striatum (P 0.001) as compared to TAT-GluN2B (10 mg per kilogram). Cortex: H(2) = 41.235; P 0.001; striatum: H(2) = 38.808; P 0.001. Kruskal-Wallis ANOVA on ranks with Dunn's post hoc; bars represent relative means ± s.e.m., ***,ΔΔΔP 0.001. n values represent tissue from 3 animals collected from at least 2 litters. Scale bars 1 mm in b,c,e, 20 μm in f. Full-length blots are presented in Supplementary Figure 9.


Figure 1Same as ( E ) with amiRhen1 - 14 scions and non - transgenic WT or hen1 - 14 rootstocks . Average relative U6 - normalized band - intensity quantifications from two biological replicates are indicated .

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Figure 1Same as (E) with amiRhen1-14 scions and non-transgenic WT or hen1-14 rootstocks. Average relative U6-normalized band-intensity quantifications from two biological replicates are indicated.


Figure 2Phenotype of pSUC2 : : amiRSUL plants in hst - 1 / - 25 / - 26 backgrounds . RT - qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes . Error - bars : SD . Welch ' s t - test p - values are indicated . n = 5 .

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Figure 2Phenotype of pSUC2::amiRSUL plants in hst-1/-25/-26 backgrounds.RT-qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes. Error-bars: SD. Welch's t-test p-values are indicated. n=5.


Figure 3amiRSUL northern analysis in input and AGO1 - immunoprecipitated ( AGO1 - IP ) fractions isolated from leaves of pSUC2 : : amiRSUL plants in the specified genotypes . No Ab : No antibody added to the HST input extract ( IP negative control ) . miR165 / 166 and U6 were probed as endogenous controls . AGO1 western analysis and Coomassie blue ( Coom . ) staining of the western blot membrane are provided as controls . amiRSUL band - intensity quantifications , U6 - normalized for input samples , are indicated .

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Figure 3amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated.


Figure 4amiRSUL northern analysis , in biological duplicates , in scions ( S ) and rootstocks ( R ) in the specified genotypes ' grafting combinations ( amiR : pSUC2 : : amiRSUL ) ( see also Appendix Fig . S12B ) . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated for each tissue . amiRSUL northern analysis , in biological duplicates , from total RNA extracted from aphids fed on WT or amiR plants , in the specified genetic backgrounds . U6 : as in ( A ) . Average relative U6 - normalized band - intensity quantifications are indicated .

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Figure 4amiRSUL northern analysis, in biological duplicates, in scions (S) and rootstocks (R) in the specified genotypes' grafting combinations (amiR: pSUC2::amiRSUL) (see also Appendix Fig.S12B). U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated for each tissue.amiRSUL northern analysis, in biological duplicates, from total RNA extracted from aphids fed on WT or amiR plants, in the specified genetic backgrounds. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.


Figure 5Localization of HST : GFP and , as a reference , of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves ( C ) or roots ( D ) . CW : calcofluor white staining . Scale bars : 20 µm . Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2 : : HST : GFP , pSUC2 : : GFP or pSUC2 : : tmGFP9 . Scale bars : 100 µm . Confocal fluorescence images of the differentiation and division zones of pSUC2 : : GFPhst - 1 or pSUC2 : : tmGFP9hst - 1 root tips . Scale bars : 100 µm .

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Figure 5Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm.Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2::HST:GFP, pSUC2::GFP or pSUC2::tmGFP9. Scale bars: 100 µm. Confocal fluorescence images of the differentiation and division zones of pSUC2::GFPhst-1 or pSUC2::tmGFP9hst-1 root tips. Scale bars: 100 µm.


Figure 6Subcellular localizations of HST : GFP expressed from the UBQ10 promoter ( pUBQ ) in Arabidopsis roots ( i ) . Distinct planes of the same root cells imaged by confocal microscopy are shown ( ii - iii ) . PI : propidium iodide staining . Scale bars : 50µm ( i ) ; 10µm ( ii - iii ) . Subcellular localizations of hst - 3 : GFP expressed from pUBQ in root cells . Distinct planes of the same root cells imaged by confocal microscopy are shown ( i - ii ) . PI : as in ( A ) . Scale bars : 10µm .

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Figure 6Subcellular localizations of HST:GFP expressed from the UBQ10 promoter (pUBQ) in Arabidopsis roots (i). Distinct planes of the same root cells imaged by confocal microscopy are shown (ii-iii). PI: propidium iodide staining. Scale bars: 50µm (i); 10µm (ii-iii).Subcellular localizations of hst-3:GFP expressed from pUBQ in root cells. Distinct planes of the same root cells imaged by confocal microscopy are shown (i-ii). PI: as in (A). Scale bars: 10µm.


Figure 7miR395 northern analysis in scions ( S ) and hyl1 - 2 rootstocks ( R ) , in the indicated genotypes ' combinations , under SO4 - sufficient ( + SO4 ) or SO4 - starved ( - SO4 ) conditions , in two biological replicates ( rep . ) . U6 was probed as an endogenous control . Relative U6 - normalized band - intensity quantifications are indicated for each tissue in - SO4 conditions . miR160 northern analysis in Meselect - separated leaf vasculature and epidermis in WT versus hst - 1 plants , in three biological replicates ( rep . ) . U6 : as in ( A ) . Relative U6 - normalized band - intensity quantifications are indicated . Basic fuchsin staining of protoxylem ( unfilled arrowheads ) and metaxylem ( filled arrowheads ) in WT , shr - 2 or hst - 1 roots . * : protoxylem gap . Scale bars : 10 µm .

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Figure 7miR395 northern analysis in scions (S) and hyl1-2 rootstocks (R), in the indicated genotypes' combinations, under SO4-sufficient (+SO4) or SO4-starved (-SO4) conditions, in two biological replicates (rep.). U6 was probed as an endogenous control. Relative U6-normalized band-intensity quantifications are indicated for each tissue in -SO4 conditions.miR160 northern analysis in Meselect-separated leaf vasculature and epidermis in WT versus hst-1 plants, in three biological replicates (rep.). U6: as in (A). Relative U6-normalized band-intensity quantifications are indicated.Basic fuchsin staining of protoxylem (unfilled arrowheads) and metaxylem (filled arrowheads) in WT, shr-2 or hst-1 roots. *: protoxylem gap. Scale bars: 10 µm.


Figure 1 ( B , Characterization of DONSON ' s chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated for the indicated times in 20 μL of interphase egg extract . Chromatin fractions were isolated and analyzed by immunoblotting . His - p27 ( 100 μg / mL , p27 ( CDKi ) ) , PHA - 767491 ( 50 μM , Cdc7i ) were supplemented as indicated . Similar experiments with ( B ) were performed in triplicate and DONSON signal intensity was quantified by Image J . C , Characterization of DONSON ' s chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated for the indicated times in 20 μL of interphase egg extract . Chromatin fractions were isolated and analyzed by immunoblotting . aphidicolin ( 10 μg / mL , aph ) , His - geminin ( 50 μg / mL , gem ) were supplemented as indicated . Similar experiments with ( C ) were performed in triplicate and DONSON signal intensity was quantified by Image J . E ) Characterization of DONSON ' s chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated for the indicated times in 20 μL of interphase egg extract . Chromatin fractions were isolated and analyzed by immunoblotting . NMS - 873 ( 100 μM , p97i ) , and MLN - 4924 ( 20 μM , Culi ) were supplemented as indicated .

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Figure 1(B, Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. His-p27 (100 μg/mL, p27 (CDKi)), PHA-767491 (50 μM, Cdc7i) were supplemented as indicated. Similar experiments with (B) were performed in triplicate and DONSON signal intensity was quantified by Image J.C, Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. aphidicolin (10 μg/mL, aph), His-geminin (50 μg/mL, gem) were supplemented as indicated. Similar experiments with (C) were performed in triplicate and DONSON signal intensity was quantified by Image J.E) Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. NMS-873 (100 μM, p97i), and MLN-4924 (20 μM, Culi) were supplemented as indicated.


Figure 2 ( A ) Immunodepletion from the interphase egg extract was performed using control IgG beads ( mock ) or anti - DONSON antibody beads ( ΔDNS ) . Recombinant Xenopus DONSON ( untagged ) was added to the mock - and DONSON - depleted extracts at 0 μM ( - ) , 0 . 06 μM ( × 1 ) , 0 . 18 ( × 3 ) , and 0 . 36 μM ( × 9 ) as indicated . The extracts ( 1 μL ) were analyzed by immunoblotting , except lanes 9 - 11 , where 0 . 4 ( . 4 ) , 0 . 2 ( . 2 ) , and 0 . 1 ( . 1 ) μL were used . Since the anti - DONSON antibody was recognized by multiple bands , the presumable DONSON band was indicated by dotted lines in a vertically expanded blot . Asterisk ( * ) , non - specific band . ( B ) DONSON requirement for the replisome assembly . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of the mock - and DONDON - depleted extracts as prepared in ( A ) for 60 min in the presence of 10 μg / mL aphidicolin . The extracts ( 0 . 5 μL ) and isolated chromatin fractions were analyzed by immunoblotting . ( C ) DONSON requirement for replisome assembly . Sperm nuclei were incubated for the indicated times and conditions in the egg extract and the chromatin fractions were analyzed by immunoblotting . Each of two types of anti - DONSON antibodies ( α - DNS # 1 , α - DNS # 2 ) was added at 14 μg / mL to suppress DONSON function .

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Figure 2(A) Immunodepletion from the interphase egg extract was performed using control IgG beads (mock) or anti-DONSON antibody beads (ΔDNS). Recombinant Xenopus DONSON (untagged) was added to the mock- and DONSON-depleted extracts at 0 μM (-), 0.06 μM (×1), 0.18 (×3), and 0.36 μM (×9) as indicated. The extracts (1 μL) were analyzed by immunoblotting, except lanes 9-11, where 0.4 (.4), 0.2 (.2), and 0.1 (.1) μL were used. Since the anti-DONSON antibody was recognized by multiple bands, the presumable DONSON band was indicated by dotted lines in a vertically expanded blot. Asterisk (*), non-specific band.(B) DONSON requirement for the replisome assembly. Sperm nuclei (5,000/μL) were incubated in 20 μL of the mock- and DONDON-depleted extracts as prepared in (A) for 60 min in the presence of 10 μg/mL aphidicolin. The extracts (0.5 μL) and isolated chromatin fractions were analyzed by immunoblotting.(C) DONSON requirement for replisome assembly. Sperm nuclei were incubated for the indicated times and conditions in the egg extract and the chromatin fractions were analyzed by immunoblotting. Each of two types of anti-DONSON antibodies (α-DNS #1, α-DNS #2) was added at 14 μg/mL to suppress DONSON function.


Figure 3 ( A ) DONSON requirement for DNA replication . Sperm nuclei were incubated for 60 min in egg extract with no IgG ( non ) , control IgG ( α - control ) , or one of the two kinds of anti - DONSON antibodies ( α - DNS # 1 , α - DNS # 2 ) in a final concentration of 14 μg / mL . Cy3 - dCTP was added to all of the reactions at 1 μΜ to visualize the DNA replication . After incubation , the nuclei were fixed , stained with Hoechst 33258 , and observed under a fluorescent microscope . Representative images are shown above . Scale bar ; 50 µm . The relative Cy3 intensity of > 100 nuclei was quantified using Image J and is shown in the box plot below . The boxes define the upper and lower quartiles ; the whiskers define max to min values ; the X - mark indicates each mean value ; the central band indicates each median ; the circles indicates outliers . P - values were calculated using the Mann - Whitney test . ns , not significant . ( B ) DONSON requirement for DNA replication . Sperm nuclei ( 5 , 000 / μL ) were incubated for 60 min in 10 μL of the untreated ( control ) , mock - ( mock ) , and DONDON - depleted ( ΔDNS ) extracts with 2 μM Cy5 - dUTP . Aphidicolin ( aph ) was added to the mock - depleted extract ( 2 . 5 μg / mL ) as a negative control . Various versions of the recombinant Xenopus DONSON ( 1 - 6 ) were added to the DONSON - depleted extracts at about 0 . 06 μM . 1 ; untagged DONSON , 2 and 3 ; GST - DONSON , 4 and 5 ; GST - DONSON ( residues 155 - 579 ) ( ΔN154 ) , 6 ; His - DONSON . In the reactions corresponding to lanes 3 and 5 , 3C protease was added to separate the GST from DONSON . After incubation , the genomic DNA was isolated and subjected to 0 . 8 % TAE agarose gel electrophoresis , followed by SYBR Gold staining . The Cy5 and SYBR Gold fluorescent signals show replicated DNA and total DNA , respectively . The same experiment was performed in triplicate ; the relative Cy5 intensities to the control reaction were quantified using Image J , as shown in the bar graph below . Error bar , mean ± standard deviation ( S . D . ) . P - values were calculated using the unpaired t - test ( two - tailed ) . ns , not significant .

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Figure 3(A) DONSON requirement for DNA replication. Sperm nuclei were incubated for 60 min in egg extract with no IgG (non), control IgG (α-control), or one of the two kinds of anti-DONSON antibodies (α-DNS #1, α-DNS #2) in a final concentration of 14 μg/mL. Cy3-dCTP was added to all of the reactions at 1 μΜ to visualize the DNA replication. After incubation, the nuclei were fixed, stained with Hoechst 33258, and observed under a fluorescent microscope. Representative images are shown above. Scale bar; 50 µm. The relative Cy3 intensity of >100 nuclei was quantified using Image J and is shown in the box plot below. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.(B) DONSON requirement for DNA replication. Sperm nuclei (5,000/μL) were incubated for 60 min in 10 μL of the untreated (control), mock- (mock), and DONDON-depleted (ΔDNS) extracts with 2 μM Cy5-dUTP. Aphidicolin (aph) was added to the mock-depleted extract (2.5 μg/mL) as a negative control. Various versions of the recombinant Xenopus DONSON (1-6) were added to the DONSON-depleted extracts at about 0.06 μM. 1; untagged DONSON, 2 and 3; GST-DONSON, 4 and 5; GST-DONSON (residues 155-579) (ΔN154), 6; His-DONSON. In the reactions corresponding to lanes 3 and 5, 3C protease was added to separate the GST from DONSON. After incubation, the genomic DNA was isolated and subjected to 0.8% TAE agarose gel electrophoresis, followed by SYBR Gold staining. The Cy5 and SYBR Gold fluorescent signals show replicated DNA and total DNA, respectively. The same experiment was performed in triplicate; the relative Cy5 intensities to the control reaction were quantified using Image J, as shown in the bar graph below. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.


Figure 4 ( A ) Salt - sensitivity of chromatin - bound DONSON . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of egg extract under each of the conditions as indicated , and the chromatin fractions were isolated with normal buffer plus 0 ( - ) , 0 . 1 , or 0 . 2 M NaCl , and analyzed by immunoblotting . In addition , 0 . 5 μL of each extract ( ext ) was analyzed for comparison . ( C ) The chromatin fractions were isolated and analyzed by immunoblotting . ( E ) ( left ) The isolated genomic DNA was analyzed in the same manner as in Figure 3B . ( right ) The same experiment was repeated six times . The relative Cy5 intensities to the control reaction ( normal α - C ) were quantified using Image J , as shown in the bar graph . Error bar , mean ± standard deviation ( S . D . ) . P - values were calculated using the unpaired t - test ( two - tailed ) . ns , not significant .

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Figure 4(A) Salt-sensitivity of chromatin-bound DONSON. Sperm nuclei (5,000/μL) were incubated in 20 μL of egg extract under each of the conditions as indicated, and the chromatin fractions were isolated with normal buffer plus 0 (-), 0.1, or 0.2 M NaCl, and analyzed by immunoblotting. In addition, 0.5 μL of each extract (ext) was analyzed for comparison.(C) The chromatin fractions were isolated and analyzed by immunoblotting.(E) (left) The isolated genomic DNA was analyzed in the same manner as in Figure 3B. (right) The same experiment was repeated six times. The relative Cy5 intensities to the control reaction (normal α-C) were quantified using Image J, as shown in the bar graph. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.


Figure 5 ( A ) Ability of DONSON deletions to support replisome assembly . The DONSON depletion experiment , as that in Figure 2B , was performed using 3 × FLAG - tagged versions of the full length ( amino acid residues 1 - 579 ) or N - terminal / C - terminal deletions ( amino acid residues 1 - 157 , 133 - 579 , 1 - 476 , 1 - 369 ) of Xenopus ( x ) DONSON , whose final concentrations were about 0 . 06 μM . The signals derived from DONSON are indicated with dotted lines . His - p27 and His - geminin were used as negative controls . Asterisk ( * ) , non - specific bands . ( B ) Interaction of DONSON with the replication factors . 3 × FLAG - tagged full length ( xF ) , N - terminal region residue 1 - 157 ( xN ) , middle region residue 133 - 369 ( xM ) , and C - terminal region residue 358 - 579 ( xC ) versions of Xenopus DONSON were added to an interphase egg extract at about 8 . 8 μM . After incubation for 15 min , the whole extract was subjected to a pull - down assay using FLAG - M2 beads , and the precipitated proteins were analyzed by immunoblotting ( FLAG - IP ) . The extracts ( 2 . 5 % ) were analyzed for comparison ( Input ) . ( D ) Identification of GINS - and Polε - interacting motifs in the N - terminal region of DONSON . The FLAG - IP experiment , as in ( B ) , was performed with 2 μM of 3 × FLAG - tagged versions of wild - type ( xN - WT ) , P6A / Y8A ( xN - 2A ) , N67A / P68A / F69A ( xN - 3A ) , and P6A / Y8A / N67A / P68A / F69A ( xN - 5A ) of Xenopus DONSON each with an N - terminal with 157 amino acids . The extracts ( 6 . 7 % ) were analyzed for comparison ( Input ) . ( E ) Interaction of human DONSON with Xenopus GINS and Polε in egg extract . The FLAG - IP experiment , as in ( B ) , was performed with 1 μM of 3 × FLAG - tagged versions of wild - type ( hF - WT ) , P6A / Y8A ( hF - 2A ) , N78A / P79A / F80A ( hF - 3A ) , P6A / Y8A / N78A / P79A / F80A ( hF - 5A ) , K489T ( hF - KT ) of full length human DONSON each as well as 3 × FLAG - tagged wild - type Xenopus DONSON ( xF - WT ) . The extracts ( 5 % ) were analyzed for comparison ( Input ) .

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

Figure 5(A) Ability of DONSON deletions to support replisome assembly. The DONSON depletion experiment, as that in Figure 2B, was performed using 3×FLAG-tagged versions of the full length (amino acid residues 1-579) or N-terminal/C-terminal deletions (amino acid residues 1-157, 133-579, 1-476, 1-369) of Xenopus (x) DONSON, whose final concentrations were about 0.06 μM. The signals derived from DONSON are indicated with dotted lines. His-p27 and His-geminin were used as negative controls. Asterisk (*), non-specific bands. (B) Interaction of DONSON with the replication factors. 3×FLAG-tagged full length (xF), N-terminal region residue 1-157 (xN), middle region residue 133-369 (xM), and C-terminal region residue 358-579 (xC) versions of Xenopus DONSON were added to an interphase egg extract at about 8.8 μM. After incubation for 15 min, the whole extract was subjected to a pull-down assay using FLAG-M2 beads, and the precipitated proteins were analyzed by immunoblotting (FLAG-IP). The extracts (2.5%) were analyzed for comparison (Input).(D) Identification of GINS- and Polε-interacting motifs in the N-terminal region of DONSON. The FLAG-IP experiment, as in (B), was performed with 2 μM of 3×FLAG-tagged versions of wild-type (xN-WT), P6A/Y8A (xN-2A), N67A/P68A/F69A (xN-3A), and P6A/Y8A/N67A/P68A/F69A (xN-5A) of Xenopus DONSON each with an N-terminal with 157 amino acids. The extracts (6.7%) were analyzed for comparison (Input). (E) Interaction of human DONSON with Xenopus GINS and Polε in egg extract. The FLAG-IP experiment, as in (B), was performed with 1 μM of 3×FLAG-tagged versions of wild-type (hF-WT), P6A/Y8A (hF-2A), N78A/P79A/F80A (hF-3A), P6A/Y8A/N78A/P79A/F80A (hF-5A), K489T (hF-KT) of full length human DONSON each as well as 3×FLAG-tagged wild-type Xenopus DONSON (xF-WT). The extracts (5%) were analyzed for comparison (Input).


Figure 6 ( A ) Requirement of GINS - and Polε - interacting motifs within DONSON for replisome assembly . This DONSON depletion experiment , as that in Figure 2B , was performed using the same recombinant proteins as in Figure 5E . Human proteins were added at 0 . 3 μM each and Xenopus protein at 0 . 06 μM . His - p27 ( p27 ) and His - geminin ( gem ) were used as negative controls . ( B ) Requirement of GINS - and Polε - interacting motifs within DONSON for replication activity . The same experiment procedure was used as in Figure 3A for the mock - and DONSON - depleted extracts with the same recombinant proteins as in Figure 5E . Xenopus proteins were added at 0 . 06 μM each while human proteins were added at 0 . 3 μM . The relative Cy3 intensity of > 100 nuclei was quantified using Image J and is shown in the box plot . The boxes define the upper and lower quartiles ; the whiskers define max to min values ; the X - mark indicates each mean value ; the central band indicates each median ; the circles indicates outliers . P - values were calculated using the Mann - Whitney test . ns , not significant .

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

Figure 6(A) Requirement of GINS- and Polε-interacting motifs within DONSON for replisome assembly. This DONSON depletion experiment, as that in Figure 2B, was performed using the same recombinant proteins as in Figure 5E. Human proteins were added at 0.3 μM each and Xenopus protein at 0.06 μM. His-p27 (p27) and His-geminin (gem) were used as negative controls.(B) Requirement of GINS- and Polε-interacting motifs within DONSON for replication activity. The same experiment procedure was used as in Figure 3A for the mock- and DONSON-depleted extracts with the same recombinant proteins as in Figure 5E. Xenopus proteins were added at 0.06 μM each while human proteins were added at 0.3 μM. The relative Cy3 intensity of >100 nuclei was quantified using Image J and is shown in the box plot. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.


Figure 7 ( A , Requirement of GINS , Cdc45 for the DONSON chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of mock - , GINS - , Cdc45 - depleted egg extract ΔGINS , ΔCdc45 for the indicated times and conditions , and the chromatin fractions were isolated and analyzed by immunoblotting . The extracts ( 0 . 5 μL ) were also analyzed . Replication proteins were categorized into three groups ( Group - 1 , - 2 , - 3 ) based on their dependency on GINS , Cdc45B ) Requirement of TopBP1 for the DONSON chromatin association . Sperm nuclei ( 5 , 000 / μL ) were incubated in 20 μL of mock - , or TopBP1 - depleted egg extract , ΔTopBP1 ) for the indicated times and conditions , and the chromatin fractions were isolated and analyzed by immunoblotting . The extracts ( 0 . 5 μL ) were also analyzed . Replication proteins were categorized into three groups ( Group - 1 , - 2 , - 3 ) based on their dependency on TopBP1 . ( C ) ( above ) Requirement of DONSON for CDK and DDK function in the replisome assembly . Sperm nuclei ( 5 , 000 / μL ) were incubated for 50 min in 120 μL of egg extract with control IgG ( α - cont ) or anti - DONSON antibody # 1 ( α - DNS ) . Each part of this first reaction ( 1st ) was divided equally into six parts , and the nuclear fractions were isolated and transferred to 20 μL of flesh extract with control IgG ( α - cont ) or anti - DONSON antibody # 1 ( α - DNS ) in the presence or absence of CDK inhibitors ( Ci : 100 μM roscovitine , 100 μg / mL His - p27 ) and Cdc7 inhibitor ( 7i : 50 μM PHA - 767491 ) . Aphidicolin was added to the first and second extracts at 10 μg / mL to suppress replication termination . After incubation for 40 min in the second reaction ( 2nd ) , the chromatin fractions were isolated and analyzed by immunoblotting . In addition , the extracts ( 0 . 5 μL ) were analyzed ( ext ) . ( below ) It is summarized whether or not replisome assembly was allowed under each condition , where DONSON , CDK , and Cdc7 were either functional ( + ) or non - functional ( - ) in the 1st and 2nd incubations .

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

Figure 7(A, Requirement of GINS, Cdc45 for the DONSON chromatin association. Sperm nuclei (5,000/μL) were incubated in 20 μL of mock-, GINS-, Cdc45- depleted egg extract ΔGINS, ΔCdc45 for the indicated times and conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. The extracts (0.5 μL) were also analyzed. Replication proteins were categorized into three groups (Group-1, -2, -3) based on their dependency on GINS, Cdc45B) Requirement of TopBP1 for the DONSON chromatin association. Sperm nuclei (5,000/μL) were incubated in 20 μL of mock-, or TopBP1- depleted egg extract , ΔTopBP1) for the indicated times and conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. The extracts (0.5 μL) were also analyzed. Replication proteins were categorized into three groups (Group-1, -2, -3) based on their dependency on TopBP1.(C) (above) Requirement of DONSON for CDK and DDK function in the replisome assembly. Sperm nuclei (5,000/μL) were incubated for 50 min in 120 μL of egg extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS). Each part of this first reaction (1st) was divided equally into six parts, and the nuclear fractions were isolated and transferred to 20 μL of flesh extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS) in the presence or absence of CDK inhibitors (Ci: 100 μM roscovitine, 100 μg/mL His-p27) and Cdc7 inhibitor (7i: 50 μM PHA-767491). Aphidicolin was added to the first and second extracts at 10 μg/mL to suppress replication termination. After incubation for 40 min in the second reaction (2nd), the chromatin fractions were isolated and analyzed by immunoblotting. In addition, the extracts (0.5 μL) were analyzed (ext). (below) It is summarized whether or not replisome assembly was allowed under each condition, where DONSON, CDK, and Cdc7 were either functional (+) or non-functional (-) in the 1st and 2nd incubations.


Figure 1A Representative images show expression of YAP in normal cervical tissues , early - stage cervical cancer ( stage I II ) , and advanced - stage cervical cancer ( stage III IV ) . Note the significant increase in YAP immunosignals ( brown staining ) in the advanced - stage tumors . Tissue core size : 1 . 5 mm . B Positivity ( percentage of positively stained cells relative to the total number of cells in the tissue section ) of YAP immunosignals in the normal and cancerous tissues . Data were analyzed with unpaired t - test in GraphPad Prism 5 with Welch ' s correction . Bars represent means ± SEM ( n = 10 for normal tissues ; n = 69 for tumor tissues ; * * * P 0 . 0001 ) . C A representative image showing the expression of YAP ( brown staining ) in normal cervical tissue . Scale bar = 20 μm . D - F Representative images showing the expression of YAP in ( D ) early - stage cervical cancer tissue ( stage Ib , T1N0M0 ) ; ( E ) medium - stage cervical cancer tissue ( stage IIb , T2N0M0 ) ; and ( F ) advanced - stage cervical cancer tissue ( stage IIIb , T3N1M0 ) . Scale bar = 20 μm . G , H High - resolution images showing the cellular location of YAP in ( G ) early - stage cervical cancer tissue and ( H ) advanced - stage cervical cancer tissue . Scale bar = 20 μm . I Multidimensional cancer genomics data analysis results showing cross - cancer YAP gene alteration . The histogram showed the frequencies of YAP gene mutation , deletion , and amplification across cancers . Data were extracted from TCGA database and analyzed using cBioPortal online analyzing tools . The results indicate that the highest alteration frequency of YAP gene occurs in cervical cancer .

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

Figure 1A Representative images show expression of YAP in normal cervical tissues, early-stage cervical cancer (stage I II), and advanced-stage cervical cancer (stage III IV). Note the significant increase in YAP immunosignals (brown staining) in the advanced-stage tumors. Tissue core size: 1.5 mm.B Positivity (percentage of positively stained cells relative to the total number of cells in the tissue section) of YAP immunosignals in the normal and cancerous tissues. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Bars represent means ± SEM (n = 10 for normal tissues; n = 69 for tumor tissues; ***P 0.0001).C A representative image showing the expression of YAP (brown staining) in normal cervical tissue. Scale bar = 20 μm.D-F Representative images showing the expression of YAP in (D) early-stage cervical cancer tissue (stage Ib, T1N0M0); (E) medium-stage cervical cancer tissue (stage IIb, T2N0M0); and (F) advanced-stage cervical cancer tissue (stage IIIb, T3N1M0). Scale bar = 20 μm.G, H High-resolution images showing the cellular location of YAP in (G) early-stage cervical cancer tissue and (H) advanced-stage cervical cancer tissue. Scale bar = 20 μm.I Multidimensional cancer genomics data analysis results showing cross-cancer YAP gene alteration. The histogram showed the frequencies of YAP gene mutation, deletion, and amplification across cancers. Data were extracted from TCGA database and analyzed using cBioPortal online analyzing tools. The results indicate that the highest alteration frequency of YAP gene occurs in cervical cancer.


Figure 2A , C , E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ ME180 - MXIV ( control ) , ME180 - YAP , and ME180 - YAPS127A cells ] ( A ) ; HT3 cell lines [ HT3 - MXIV ( control ) , HT3 - YAP , and HT3 - YAPS127A cells ] ( C ) ; and Ect1 cell lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , and Ect1 - YAPS127A cells ] ( E ) . β - Actin was used as a protein loading control . B , D , F Growth curves of YAP - overexpressed ME180 cell lines [ ME180 - MXIV ( control ) , ME180 - YAP , and ME180 - YAPS127A cells ] ( B ) ; HT3 cell lines [ HT3 - MXIV ( control ) , HT3 - YAP , and HT3 - YAPS127A cells ] ( D ) ; and Ect1 cell lines [ Ect1 - MXIV ( control ) , Ect1 - YAP , and Ect1 - YAPS127A cells ] ( F ) . Each point represents the mean ± SEM ( n = 4 ) . * * * P < 0 . 0001 , ME180 - MXIV vs . ME180 - YAP cells and ME180 - MXIV vs . ME180 - YAPS127A cells on day 8 . * * * P < 0 . 0001 , HT3 - MXIV vs . HT3 - YAP cells and HT3 - MXIV vs . HT3 - YAPS127A cells on day 8 . * * * P < 0 . 0001 , Ect1 - MXIV vs . Ect1 - Ect1 - YAPS127A cells on day 4 . * P < 0 . 0074 , Ect1 - MXIV vs . Ect1 - YAP cells on day 4 . G Western blot showing YAP levels in non - targeting control siRNA ( siCtrl ) - and YAP siRNA ( siYAP ) - transfected HT3 cells . H Proliferation of HT3 cells treated with control ( siCtrl ) or YAP siRNA ( siYAP ) . Each point represents the mean ± SEM ( n = 5 ) . * * * P < 0 . 001 compared with siCtrl ( siCtrl vs . siYAP , P = 0 . 0002 ) .

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

Figure 2A, C, E Western blot analysis showing levels of YAP and phosphorylated YAP in ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (A); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (C); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (E). β-Actin was used as a protein loading control.B, D, F Growth curves of YAP-overexpressed ME180 cell lines [ME180-MXIV (control), ME180-YAP, and ME180-YAPS127A cells] (B); HT3 cell lines [HT3-MXIV (control), HT3-YAP, and HT3-YAPS127A cells] (D); and Ect1 cell lines [Ect1-MXIV (control), Ect1-YAP, and Ect1-YAPS127A cells] (F). Each point represents the mean ± SEM (n = 4). ***P < 0.0001, ME180-MXIV vs. ME180-YAP cells and ME180-MXIV vs. ME180-YAPS127A cells on day 8. ***P < 0.0001, HT3-MXIV vs. HT3-YAP cells and HT3-MXIV vs. HT3-YAPS127A cells on day 8. ***P < 0.0001, Ect1-MXIV vs. Ect1-Ect1-YAPS127A cells on day 4. *P < 0.0074, Ect1-MXIV vs. Ect1-YAP cells on day 4.G Western blot showing YAP levels in non-targeting control siRNA (siCtrl)- and YAP siRNA (siYAP)-transfected HT3 cells.H Proliferation of HT3 cells treated with control (siCtrl) or YAP siRNA (siYAP). Each point represents the mean ± SEM (n = 5). ***P < 0.001 compared with siCtrl (siCtrl vs. siYAP, P = 0.0002).


Figure 3A Representative images showing colonies formed by Ect1 - MXIV , Ect1 - YAP , and Ect1 - YAPS127A cells after growth in soft agar for 9 days . Scale bar : 500 μm . B Quantitative data showing colony formation in Ect1 - MXIV , Ect1 - YAP , and Ect1 - YAPS127A cells . Each bar represents mean ± SEM of five independent experiments . Bars with different letters are significantly different from each other ( Ect1 - MXIV vs . Ect1 - YAP , P = 0 . 0003 ; Ect1 - MXIV vs . Ect1 - YAPS127A , P = 0 . 0002 ) . C , E Representative images showing the anchorage - independent growth of YAP - expressing HT3 ( C ) and ME180 ( E ) cervical cancer cell lines . Anchorage - independent cell growth was determined by the soft agar colony formation assay . Scale bar : 500 μm . D , F Fluorescence - based quantitative analysis showing the differences of anchorage - independent growth in HT3 - MXIV , HT3 - YAP , and HT3 - YAPS127A ( D ) and ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells ( F ) . The anchorage - independent cell growth was determined using a CytoSelect 96 - Well Cell Transformation Assay kit , and data are presented as relative fluorescent units ( RFU ) . Each bar represents mean ± SEM of four independent experiments . Bars with different letters are significantly different from each other ( HT3 - MXIV vs . HT3 - YAP , P = 0 . 0002 ; HT3 - MXIV vs . HT3 - YAPS127A , P < 0 . 0001 ; ME - MXIV vs . ME - YAP , P = 0 . 0011 ; ME - MXIV vs . ME - YAPS127A , P = 0 . 0013 ) .

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

Figure 3A Representative images showing colonies formed by Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells after growth in soft agar for 9 days. Scale bar: 500 μm.B Quantitative data showing colony formation in Ect1-MXIV, Ect1-YAP, and Ect1-YAPS127A cells. Each bar represents mean ± SEM of five independent experiments. Bars with different letters are significantly different from each other (Ect1-MXIV vs. Ect1-YAP, P = 0.0003; Ect1-MXIV vs. Ect1-YAPS127A, P = 0.0002).C, E Representative images showing the anchorage-independent growth of YAP-expressing HT3 (C) and ME180 (E) cervical cancer cell lines. Anchorage-independent cell growth was determined by the soft agar colony formation assay. Scale bar: 500 μm.D, F Fluorescence-based quantitative analysis showing the differences of anchorage-independent growth in HT3-MXIV, HT3-YAP, and HT3-YAPS127A (D) and ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells (F). The anchorage-independent cell growth was determined using a CytoSelect 96-Well Cell Transformation Assay kit, and data are presented as relative fluorescent units (RFU). Each bar represents mean ± SEM of four independent experiments. Bars with different letters are significantly different from each other (HT3-MXIV vs. HT3-YAP, P = 0.0002; HT3-MXIV vs. HT3-YAPS127A, P < 0.0001; ME-MXIV vs. ME-YAP, P = 0.0011; ME-MXIV vs. ME-YAPS127A, P = 0.0013).


Figure 4A , B Representative images of tumorxenografts of ME180 - MXIV ( MX , right flank ) , ME180 - YAP ( A ) ( YAP , left flank ) , and ME180 - YAPS127A ( B ) ( YAPS127A , left flank ) cells implanted in athymic nude mice . Red lines indicate the edge of tumors . C The growth curve of humantumorxenografts derived from control ( ME - MX ) and YAP - overexpressing ME180cervical cancer cell lines ( ME - YAP , ME - YAPS127A ) implanted in the athymic nude mice . Each point represents mean ± SEM of six tumors ( control n = 10 ) . D Representative images showing the relative size of tumors from control and YAP - overexpressing ME180 cervical cancer cells . E The average weight of tumor xenografts from the control and YAP - overexpressing ME180 cell lines . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc tests . Each bar represents the mean ± SEM ( n = 10 for control , n = 6 for YAP and YAPS127A groups ) . Bars with different letters are significantly different from each other ( MXIV vs . YAP , P = 0 . 0008 ; MXIV vs . YAPS127A , P < 0 . 0001 ) . F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells . G Expression of Ki67 in the tumor xenografts of ME180 - MXIV , ME180 - YAP , and ME180 - YAPS127A cells implanted in athymic nude mice . Ki67 was visualized using an Alexa - 488 ( green ) - conjugated secondary antibody . Actin was visualized using an Alexa - 594 ( red ) - conjugated secondary antibody . Nuclei were stained with DAPI . Scale bar : 20 μm . Note the significant increase in the Ki67 - positive cells in the ME180 - YAP and ME180 - YAPS127A tumor xenografts . H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector ( shCtrl ) or lentivirus - based YAP shRNAs ( shYAP # 1 or shYAP # 2 ) . I Representative images showing tumor xenografts derived from ME180 - shCtrl cells ( left flank ) and ME180 - shYAP # 1 cells ( right flank ) ( n = 6 ) . J Representative images showing the relative size of tumors derived from ME180 cervical cancer cells transfected with control shRNA ( left , shCtrl ) or YAP shRNA # 1 ( right , shYAP # 1 ) ( n = 6 ) .

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

Figure 4A, B Representative images of tumorxenografts of ME180-MXIV (MX, right flank), ME180-YAP (A) (YAP, left flank), and ME180-YAPS127A (B) (YAPS127A, left flank) cells implanted in athymic nude mice. Red lines indicate the edge of tumors.C The growth curve of humantumorxenografts derived from control (ME-MX) and YAP-overexpressing ME180cervical cancer cell lines (ME-YAP, ME-YAPS127A) implanted in the athymic nude mice. Each point represents mean ± SEM of six tumors (control n = 10).D Representative images showing the relative size of tumors from control and YAP-overexpressing ME180 cervical cancer cells.E The average weight of tumor xenografts from the control and YAP-overexpressing ME180 cell lines. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents the mean ± SEM (n = 10 for control, n = 6 for YAP and YAPS127A groups). Bars with different letters are significantly different from each other (MXIV vs. YAP, P = 0.0008; MXIV vs. YAPS127A, P < 0.0001).F Western blot analysis of YAP protein levels in the tumor xenografts derived from ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells.G Expression of Ki67 in the tumor xenografts of ME180-MXIV, ME180-YAP, and ME180-YAPS127A cells implanted in athymic nude mice. Ki67 was visualized using an Alexa-488 (green)-conjugated secondary antibody. Actin was visualized using an Alexa-594 (red)-conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 20 μm. Note the significant increase in the Ki67-positive cells in the ME180-YAP and ME180-YAPS127A tumor xenografts.H Western blot analysis showing YAP protein levels in ME180 cells transfected with lentiviral empty vector (shCtrl) or lentivirus-based YAP shRNAs (shYAP#1 or shYAP#2).I Representative images showing tumor xenografts derived from ME180-shCtrl cells (left flank) and ME180-shYAP#1 cells (right flank) (n = 6).J Representative images showing the relative size of tumors derived from ME180 cervical cancer cells transfected with control shRNA (left, shCtrl) or YAP shRNA#1 (right, shYAP#1) (n = 6).