The correlation of LSD1 R838me2a with LSD1 ( E ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al .

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The correlation of LSD1 R838me2a with LSD1 (E) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.


F , G The correlation of , CARM1 with LSD1 R838me2a ( F ) and CARM1 with LSD1 ( G ) was statistically significant among breast cancer specimens ( n = 70 breast tumor samples ; E , R2 = 0 . 8835 , P < 0 . 0001 ; F , R2 = 0 . 889 , P < 0 . 0001 ; G , R2 = 0 . 8478 , P < 0 . 0001 ; Pearson correlation test ) . Note that some samples have overlapping scores . IHC score was evaluated according to Feng et al .

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F, G The correlation of , CARM1 with LSD1 R838me2a (F) and CARM1 with LSD1 (G) was statistically significant among breast cancer specimens (n = 70 breast tumor samples; E, R2 = 0.8835, P < 0.0001; F, R2 = 0.889, P < 0.0001; G, R2 = 0.8478, P < 0.0001; Pearson correlation test). Note that some samples have overlapping scores. IHC score was evaluated according to Feng et al.


A . Volcano blot representation of the differently expressed genes in FCRLS - and CD11b - positive Grn - / - microglia in comparison to wt microglia isolated from 5 . 5 - month - old mice ( male , n = 5 ) . 116 genes out of 529 genes analyzed , are significantly changed , and from these 58 genes are up - and 58 genes are down regulated . Eight genes with the highest fold change are highlighted .

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A. Volcano blot representation of the differently expressed genes in FCRLS- and CD11b- positive Grn-/- microglia in comparison to wt microglia isolated from 5.5-month-old mice (male, n = 5). 116 genes out of 529 genes analyzed, are significantly changed, and from these 58 genes are up- and 58 genes are down regulated. Eight genes with the highest fold change are highlighted.


B . Heatmap of significantly affected genes ( p < 0 . 05 ) in FCRLS - and CD11b - positive Grn - / - microglia in comparison to Grn + / + microglia isolated from 5 . 5 - month - old mice ( n = 5 per genotype ) . For the significantly affected genes of the Grn - / - microglia , mRNA expression data of the Trem2 - / - microglia in comparison to the corresponding wt microglia were taken from previously published data ( Mazaheri et al , 2017 ) . The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation ( n ≥ 5 per genotype ) . * labelled genes were not analyzed or below detection limit in Trem2 - / - microglia mRNA expression data set ( Mazaheri et al , 2017 ) .

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B. Heatmap of significantly affected genes (p < 0.05) in FCRLS- and CD11b-positive Grn-/- microglia in comparison to Grn+/+ microglia isolated from 5.5-month-old mice (n = 5 per genotype). For the significantly affected genes of the Grn-/- microglia, mRNA expression data of the Trem2-/- microglia in comparison to the corresponding wt microglia were taken from previously published data (Mazaheri et al, 2017). The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation (n ≥ 5 per genotype). * labelled genes were not analyzed or below detection limit in Trem2-/- microglia mRNA expression data set (Mazaheri et al, 2017).


D . Gene expression levels of FCRLS - and CD11b - positive Grn - / - microglia normalized to wt in comparison to the published normalized data set of Trem2 - / - microglia ( Mazaheri et al , 2017 ) .

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D. Gene expression levels of FCRLS- and CD11b-positive Grn-/- microglia normalized to wt in comparison to the published normalized data set of Trem2-/- microglia (Mazaheri et al, 2017).


A . Immunoblot analysis of ApoE , CLEC7A , TREM2 and CD68 in lysates of acutely isolated microglia from Grn - / - and Grn + / + mice ( 9 months of age , n = 3 per genotype , female ) . IBA1 was used as loading control .

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A. Immunoblot analysis of ApoE, CLEC7A, TREM2 and CD68 in lysates of acutely isolated microglia from Grn-/- and Grn+/+ mice (9 months of age, n=3 per genotype, female). IBA1 was used as loading control.


B . Quantification of protein expression normalized to levels of Grn + / + microglia ( n = 3 ) . Data represent the mean ± SD .

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B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n=3). Data represent the mean ± SD.


C . ELISA mediated quantification of sTREM2 in brain homogenates of Grn - / - and Grn + / + mice Data are shown as mean ± SEM ( n = 3 - 6 ) . D . ELISA mediated quantification of sTREM2 in serum of Grn - / - and Grn + / + mice . Data are shown as mean ± SEM ( n = 4 - 13 ) .

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C. ELISA mediated quantification of sTREM2 in brain homogenates of Grn-/- and Grn+/+ mice Data are shown as mean ± SEM (n=3-6). D. ELISA mediated quantification of sTREM2 in serum of Grn-/- and Grn+/+ mice. Data are shown as mean ± SEM (n=4-13).


E . Microglial expression of P2RY12 in cortical sections of wt , Grn - / - and Trem2 - / - mice . ( 9 months of age , female ) F . Quantification of P2RY12 positive microglia . Data are shown as mean ± SD ( n = 3 per genotype , female , except one male for Trem2 - / - ) .

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E. Microglial expression of P2RY12 in cortical sections of wt, Grn-/-and Trem2-/- mice. (9 months of age, female) F. Quantification of P2RY12 positive microglia. Data are shown as mean ± SD (n=3 per genotype, female, except one male for Trem2-/-).


G . Immunoblot analysis of secreted PGRN and ApoE ( ApoEmed ) in conditioned media and PGRN , ApoE , CLEC7A , P2RY12 and TREM2 in lysates of BV2 wild type ( Grnwt ) and PGRN deficient ( Grnmut ) cells . Soluble amyloid precursor protein ( APPs ) and calnexin were used as a loading control .

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G. Immunoblot analysis of secreted PGRN and ApoE (ApoEmed) in conditioned media and PGRN, ApoE, CLEC7A, P2RY12 and TREM2 in lysates of BV2 wild type (Grnwt) and PGRN deficient (Grnmut) cells. Soluble amyloid precursor protein (APPs) and calnexin were used as a loading control.


H . Quantification of Immunoblots normalized to BV2 Grnwt levels ( n = 3 - 5 ) . Data represent the mean ± SD .

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H. Quantification of Immunoblots normalized to BV2 Grnwt levels (n=3-5). Data represent the mean ± SD.


I . ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells . Data represent the mean ±

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I. ELISA mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SD


J . Quantification of relative mRNA levels of Apoe , Clec7a , P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells . Data represent the mean ± SD .

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J. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12 and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD.


A . Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E . coli as target particles . Phagocytosis was terminated after 30 and 60 minutes ( min ) of incubation . Data are presented as mean percentage of cells positive for pHrodo uptake ± S . D ( n = 3 ) .

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A. Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 minutes (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo uptake ± S.D (n=3).


B . Enhanced migration of CD68 positive , PGRN deficient cells in an ex vivo model . Immunofluorescence analysis of cultured Grn + / + and Grn - / - organotypic brain at 7 DIV immunostained using microglial marker CD68 . Nuclei were counterstained using DAPI . Images of boxed regions in left panels are shown at higher magnifications in right panels . Scale bars : 500 µm ( left panels ) , 250 µm ( right panels ) . Quantitative analysis reveals that Grn - / - microglia migrate larger distances than Grn + / + . Data are shown as mean ± SD ( n = 3 independent experiments ) Quantification displays an increased number of migrating Grn - / - cells compared to Grn + / + cells . Data are shown as mean ± SD ( n = 3 independent experiments )

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B. Enhanced migration of CD68 positive, PGRN deficient cells in an ex vivo model. Immunofluorescence analysis of cultured Grn+/+ and Grn-/- organotypic brain at 7 DIV immunostained using microglial marker CD68. Nuclei were counterstained using DAPI. Images of boxed regions in left panels are shown at higher magnifications in right panels. Scale bars: 500 µm (left panels), 250 µm (right panels). Quantitative analysis reveals that Grn-/- microglia migrate larger distances than Grn+/+. Data are shown as mean ± SD (n=3 independent experiments) Quantification displays an increased number of migrating Grn-/- cells compared to Grn+/+ cells. Data are shown as mean ± SD (n=3 independent experiments)


E . Enhanced microglial clustering around amyloid plaques in the absence of PGRN . Left : IBA1 - stained microglia clustering around X - 34 - positive amyloid plaque cores in 4 - month - old APPPS1 / Grn + / + mice . Right : Age - matched APPPS1 / Grn - / - mice display microglial hyper - clustering around amyloid plaques compared to APPPS1 / Grn + / + . Dotted white boxes indicate the area that is shown at higher magnification . Scale bars indicate 10µm and 3µm in inset . Immunohistochemical quantification of IBA1 positive microglial cell number per

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E. Enhanced microglial clustering around amyloid plaques in the absence of PGRN. Left: IBA1-stained microglia clustering around X-34-positive amyloid plaque cores in 4-month-old APPPS1/Grn+/+ mice. Right: Age-matched APPPS1/Grn-/- mice display microglial hyper-clustering around amyloid plaques compared to APPPS1/Grn+/+. Dotted white boxes indicate the area that is shown at higher magnification. Scale bars indicate 10µm and 3µm in inset. Immunohistochemical quantification of IBA1 positive microglial cell number per plaqu


A . TSPO - µPET in rodents : Axial slices ( A1 , A2 , A3 ) of averaged % - TSPO - PET differences between Grn - / - or Trem2 - / - mice and wt indicate increased microglial activity in the cerebrum of Grn - / - mice ( hot color scale ) and decreased microglial activity in the cerebrum of Trem2 - / - mice ( cold color scale ) . Scatter plot depicts single TSPO - µPET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 8 at 11 months ; wt n = 10 at 8 - 11 months ) . Statistics derive from one - way ANOVA with Tukey post - hoc test .

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A. TSPO-µPET in rodents: Axial slices (A1, A2, A3) of averaged %-TSPO-PET differences between Grn-/-or Trem2-/- mice and wt indicate increased microglial activity in the cerebrum of Grn-/- mice (hot color scale) and decreased microglial activity in the cerebrum of Trem2-/- mice (cold color scale). Scatter plot depicts single TSPO-µPET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=8 at 11 months; wt n=10 at 8-11 months). Statistics derive from one-way ANOVA with Tukey post-hoc test.


B . PGRN serum levels of the patients subjected to TSPO - PET confirmed the GRN mutation and non - GRN mutation carrier . PGRN was measured by ELISA and normalized to serum levels of a healthy control .

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B. PGRN serum levels of the patients subjected to TSPO-PET confirmed the GRN mutation and non-GRN mutation carrier. PGRN was measured by ELISA and normalized to serum levels of a healthy control.


C . TSPO - PET in human FTLD patients : Visual interpretation of axial TSPO - PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices ( white arrows ) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation . PET results are illustrated upon an MRI template ; 60 - 80 min p . i . emission recording ; global mean scaling . Both patients were low - affinity binder .

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C. TSPO-PET in human FTLD patients: Visual interpretation of axial TSPO-PET SUVR images in upper cranial layers reveals increased microglial activity in frontal and parietal cortices (white arrows) of a FTLD patient with a GRN loss of function mutation in comparison to a FTLD patient without a GRN mutation. PET results are illustrated upon an MRI template; 60-80 min p.i. emission recording; global mean scaling. Both patients were low-affinity binder.


FDG - µPET in rodents : Axial slices ( as indicated in Fig . 4A ) of averaged % - FDG - µPET differences between Grn - / - or Trem2 - / - mice and wt indicate decreased glucose metabolism in the cerebrum of Grn - / - and Trem2 - / - ( both cold color scale ) when compared to wt . Scatter plot depicts single FDG - PET values deriving from a neocortical volume - of - interest . µPET results are illustrated upon an MRI template ; 6 - 10 female mice per group ( Grn - / - n = 6 at 8 months , Trem2 - / - n = 6 at 11 months ; wt n = 10 at 8 - 11 months ) .

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FDG-µPET in rodents: Axial slices (as indicated in Fig. 4A) of averaged %-FDG-µPET differences between Grn-/-or Trem2-/- mice and wt indicate decreased glucose metabolism in the cerebrum of Grn-/- and Trem2-/- (both cold color scale) when compared to wt. Scatter plot depicts single FDG-PET values deriving from a neocortical volume-of-interest. µPET results are illustrated upon an MRI template; 6-10 female mice per group (Grn-/- n=6 at 8 months, Trem2-/- n=6 at 11 months; wt n=10 at 8-11 months).


A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( A ) : 200 μm .

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A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (A): 200 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell migration ( B ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( B ) : 100 μm .

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A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to transwell migration (B) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (B): 100 μm.


( C ) Effect of WDR63 knockdown on the migration of A549 cells was analyzed by single cell tracking with time - lapse microscopy at 10 min intervals for 4 h . Scale bar : 200 μm . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA .

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(C) Effect of WDR63 knockdown on the migration of A549 cells was analyzed by single cell tracking with time-lapse microscopy at 10 min intervals for 4 h. Scale bar: 200 μm. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA.


( D ) A549 cells expressing control shRNA , WDR63 shRNA # 1 , WDR63 shRNA # 2 , PCDH control , or PCDH - Flag - WDR63 were subjected to transwell invasion assay . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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(D) A549 cells expressing control shRNA, WDR63 shRNA#1, WDR63 shRNA#2, PCDH control, or PCDH-Flag-WDR63 were subjected to transwell invasion assay. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell migration ( E ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to transwell migration (E) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar : 100 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to single cell tracking ( F ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar 200 μm .

A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to single cell tracking (F) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar 200 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 1 plus shRNA - resistant Flag - WDR63 were subjected to transwell invasion ( G ) assays . The shown images are representative of three independent experiments . Cell migration speed , persistence , and mean squared displacement ( MSD ) were also analyzed using an Excel macro described by Gorelik and Gautreau . Data shown are mean ± SD . n = 3 independent experiments . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#1 plus shRNA-resistant Flag-WDR63 were subjected to transwell invasion (G) assays. The shown images are representative of three independent experiments. Cell migration speed, persistence, and mean squared displacement (MSD) were also analyzed using an Excel macro described by Gorelik and Gautreau. Data shown are mean ± SD. n = 3 independent experiments. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar : 100 μm.


( H ) A549 cells expressing control shRNA , WDR63 shRNA # 1 , or WDR63 shRNA # 2 ( each also expressing luciferase ) were injected via tail vein into nude mice ( n = 6 for each group ) . Three weeks after injection , tumor formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

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

(H) A549 cells expressing control shRNA, WDR63 shRNA#1, or WDR63 shRNA#2 (each also expressing luciferase) were injected via tail vein into nude mice (n=6 for each group). Three weeks after injection, tumor formation was monitored by bioluminescence imaging. Data shown are mean ± SD; *, P ˂ 0.05; One-way ANOVA.


( A ) WDR63 mRNA and protein levels in A549 , H292 , and HCT116 cells transduced with empty vector ( PCDH ) or PCDH - p53 . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(A) WDR63 mRNA and protein levels in A549, H292, and HCT116 cells transduced with empty vector (PCDH) or PCDH-p53. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; ***, P ˂ 0.001; two-tailed Student's t-test.


( B ) WDR63 mRNA and protein levels in A549 , H292 , and HCT116 cells transduced with control ( shctrl ) or p53 ( shp53 ) shRNA . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(B) WDR63 mRNA and protein levels in A549, H292, and HCT116 cells transduced with control (shctrl) or p53 (shp53) shRNA. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( C ) A549 cells expressing control or p53 shRNA were treated with doxorubicin ( Dox , 1μg / ml ) for the indicated periods of time . WDR63 mRNA and protein levels were then examined . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(C) A549 cells expressing control or p53 shRNA were treated with doxorubicin (Dox, 1μg/ml) for the indicated periods of time. WDR63 mRNA and protein levels were then examined. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( D ) WDR63 mRNA levels in H1299 cells transfected with empty vector ( - ) , wild - type p53 , or the indicated p53 mutants . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(D) WDR63 mRNA levels in H1299 cells transfected with empty vector (-), wild-type p53, or the indicated p53 mutants. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( F ) Lysates from A549 cells were subjected to ChIP assay using control IgG or anti - p53 antibody . ChIP products were amplified by real - time PCR . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(F) Lysates from A549 cells were subjected to ChIP assay using control IgG or anti-p53 antibody. ChIP products were amplified by real-time PCR. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test.


( G ) A549 cells were transfected with empty vector ( ctrl ) or Flag - p53 plus the indicated reporter constructs . Twenty - four hours later , reporter activity was measured . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test .

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

(G) A549 cells were transfected with empty vector (ctrl) or Flag-p53 plus the indicated reporter constructs. Twenty-four hours later, reporter activity was measured. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; ***, P ˂ 0.001; two-tailed Student's t-test.


( H ) A549 cells expressing control or p53 shRNA were transfected with pGL3 - BS1 and Renilla luciferase plasmids . Twenty - four hours later , cells were treated with or without Nutlin ( 10 μM ) for another 12 h . Reporter activity was then measured . Data shown are mean ± SD ; n = 3 independent experiments . * * , P ˂ 0 . 01 ; two - tailed Student ' s t - test .

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

(H) A549 cells expressing control or p53 shRNA were transfected with pGL3-BS1 and Renilla luciferase plasmids. Twenty-four hours later, cells were treated with or without Nutlin (10 μM) for another 12 h. Reporter activity was then measured. Data shown are mean ± SD; n = 3 independent experiments. **, P ˂ 0.01; two-tailed Student's t-test.


( A ) A ~ 44 kD protein band specifically pulled down by GST - WDR63 from A549 cell lysates was identified as Arp2 by mass spectrometry analysis . The Arp2 peptide sequences obtained by MS are shown .

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

(A) A ~44 kD protein band specifically pulled down by GST-WDR63 from A549 cell lysates was identified as Arp2 by mass spectrometry analysis. The Arp2 peptide sequences obtained by MS are shown.


( B ) Lysates from HEK293T cells expressing HA - Arp2 alone or together with Flag - WDR63 were immunoprecipitated by anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments .

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

(B) Lysates from HEK293T cells expressing HA-Arp2 alone or together with Flag-WDR63 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.


( C ) Lysates from HEK293T cells expressing GFP - WDR63 alone or together with Flag - Arp2 were immunoprecipitated by anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments .

(C) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with Flag-Arp2 were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.


( D ) Lysates from A549 cells were immunoprecipitated with control IgG or anti - WDR63 antibody , followed by western blot analysis . Data shown represent three independent experiments .

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

(D) Lysates from A549 cells were immunoprecipitated with control IgG or anti-WDR63 antibody, followed by western blot analysis. Data shown represent three independent experiments.


( E ) Purified GST or GST - Arp2 proteins immobilized on glutathione beads were incubated with purified Flag - WDR63 . Input and bead - bound proteins were analyzed by western blot . Data shown represent three independent experiments .

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

(E) Purified GST or GST-Arp2 proteins immobilized on glutathione beads were incubated with purified Flag-WDR63. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.


( F ) Purified GST or GST - WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2 . Input and bead - bound proteins were analyzed by western blot . Data shown represent three independent experiments .

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

(F) Purified GST or GST-WDR63 proteins immobilized on glutathione beads were incubated with purified Arp2. Input and bead-bound proteins were analyzed by western blot. Data shown represent three independent experiments.


( G ) Lysates from HEK293T cells expressing GFP - WDR63 alone or together with the indicated Flag - tagged each single subunit of the Arp2 / 3 complex were immunoprecipitated with anti - Flag antibody , followed by western blot analysis . Data shown represent three independent experiments .

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

(G) Lysates from HEK293T cells expressing GFP-WDR63 alone or together with the indicated Flag-tagged each single subunit of the Arp2/3 complex were immunoprecipitated with anti-Flag antibody, followed by western blot analysis. Data shown represent three independent experiments.


( H ) Purified Flag - tagged full - length or the indicated truncated WDR63 proteins immobilized on anti - Flag M2 beads were incubated with the purified Arp2 / 3 complex ( Cytoskeleton , Inc . ) in vitro . Input and beads - bound proteins were analyzed by western blot . Data shown represent three independent experiments .

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

(H) Purified Flag-tagged full-length or the indicated truncated WDR63 proteins immobilized on anti-Flag M2 beads were incubated with the purified Arp2/3 complex (Cytoskeleton, Inc.) in vitro. Input and beads-bound proteins were analyzed by western blot. Data shown represent three independent experiments.


( A ) Immunolocalization of Arp2 in a wound - healing assay . A549 cells expressing control or WDR63 were serum - starved for 6 h before they were scratched . Cells were then treated with or without 10 % FBS for 30 min , followed by immunofluorescence assay . The branched actin networks were shown by immunostaining with anti - Cortactin antibody . Data shown represent three independent experiments . Scale bar : 20 μm . The lamellipodia formation was quantified by measuring the length of the outer margin of lamellipodia in individual cell ( n = 20 for each condition ) , and expressed as the proportion of the total length of the cell perimeter . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; One - way ANOVA .

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

(A) Immunolocalization of Arp2 in a wound-healing assay. A549 cells expressing control or WDR63 were serum-starved for 6 h before they were scratched. Cells were then treated with or without 10% FBS for 30 min, followed by immunofluorescence assay. The branched actin networks were shown by immunostaining with anti-Cortactin antibody. Data shown represent three independent experiments. Scale bar: 20 μm. The lamellipodia formation was quantified by measuring the length of the outer margin of lamellipodia in individual cell (n=20 for each condition), and expressed as the proportion of the total length of the cell perimeter. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; One-way ANOVA.


( B and C ) Effects of full - length WDR63 ( B ) and WDR63 ( 1 - 200 ) ( C ) on Arp2 / 3 - mediated actin polymerization were examined by pyrene actin polymerization assays . Various concentrations of WDR63 ( B ) or WDR63 ( 1 - 200 ) ( C ) were incubated with 2 μM actin ( 50 % pyrene - labeled ) , 10 nM Arp2 / 3 and 400 nM GST - VCA as indicated . Data shown represent three independent experiments .

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

(B and C) Effects of full-length WDR63 (B) and WDR63 (1-200) (C) on Arp2/3-mediated actin polymerization were examined by pyrene actin polymerization assays. Various concentrations of WDR63 (B) or WDR63 (1-200) (C) were incubated with 2 μM actin (50% pyrene-labeled), 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Data shown represent three independent experiments.


( D ) Effect of WDR63 on Arp2 / 3 - mediated branched actin polymerization was analyzed by electron microscopy . 0 . 6 μM WDR63 was incubated with 2 μM actin , 10 nM Arp2 / 3 and 400 nM GST - VCA as indicated . Actin structures were then stabilized with addition of 2μM phalloidin , followed by electron microscopy analysis . The arrows indicate actin branch junctions . The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments . For each condition , over 150 actin filaments were counted . Data shown are mean ± SD ; n = 3 independent experiments . * * * , P ˂ 0 . 001 ; two - tailed Student ' s t - test . Scale bar : 200 nm .

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

(D) Effect of WDR63 on Arp2/3-mediated branched actin polymerization was analyzed by electron microscopy. 0.6 μM WDR63 was incubated with 2 μM actin, 10 nM Arp2/3 and 400 nM GST-VCA as indicated. Actin structures were then stabilized with addition of 2μM phalloidin, followed by electron microscopy analysis. The arrows indicate actin branch junctions. The percentage of branched actin filaments was expressed as the ratio of the number of actin filaments with branch junctions to the total numbers of actin filaments. For each condition, over 150 actin filaments were counted. Data shown are mean ± SD; n = 3 independent experiments. ***, P ˂ 0.001; two-tailed Student's t-test. Scale bar: 200 nm.


( E and F ) Increasing amounts of purified Flag - WDR63 ( 0 . 1 μM , 0 . 2 μM , and 0 . 4 μM ) ( E ) or Flag - WDR63 ( 1 - 200 ) ( 0 . 25 μM , 0 . 5 μM , and 1 μM ) ( F ) was incubated with the Arp2 / 3 complex ( 10 nM ) and GST - VCA as indicated in vitro , followed by GST pull - down assay . Data shown represent three independent experiments .

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

(E and F) Increasing amounts of purified Flag-WDR63 (0.1 μM, 0.2 μM, and 0.4 μM) (E) or Flag-WDR63 (1-200) (0.25 μM, 0.5 μM, and 1 μM) (F) was incubated with the Arp2/3 complex (10 nM) and GST-VCA as indicated in vitro, followed by GST pull-down assay. Data shown represent three independent experiments.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bar in ( A ) : 200 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bar in (A): 200 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA Lysates from these cells were also analyzed by western blot ( B ) . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA Lysates from these cells were also analyzed by western blot (B). Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to transwell migration ( C ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bars in ( C : 100 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell migration (C) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars in (C : 100 μm.


A549 cells expressing control shRNA , WDR63 shRNA # 1 , Arp2 shRNA , or both WDR63 and Arp2 shRNA were subjected to transwell invasion ( D ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; N . S . , no significance ; One - way ANOVA . Scale bars 100 μm .

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

A549 cells expressing control shRNA, WDR63 shRNA#1, Arp2 shRNA, or both WDR63 and Arp2 shRNA were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; N.S., no significance; One-way ANOVA. Scale bars 100 μm.


A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell migration ( E ) The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Lysates from these cells were also analyzed by western blot ( F ) . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA .

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

A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Lysates from these cells were also analyzed by western blot (F). Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA.


A549 cells expressing either control shRNA or WDR63 shRNA # 1 were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell invasion ( G ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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

A549 cells expressing either control shRNA or WDR63 shRNA#1 were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (G) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to wound - healing ( A ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar in ( A ) : 200 μm .

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

A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to wound-healing (A) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bar in (A): 200 μm.


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 Lysates from these cells were also analyzed by western blot ( B ) .

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

A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 Lysates from these cells were also analyzed by western blot (B).


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell migration ( C ) The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bars 100 μm .

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A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell migration (C) The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm.


A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 were subjected to transwell invasion ( D ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bars 100 μm .

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A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 were subjected to transwell invasion (D) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). **, P ˂ 0.01; ***, P ˂ 0.001; One-way ANOVA. Scale bars 100 μm.


A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell migration ( E ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

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A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell migration (E) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag - tagged full - length or truncated WDR63 proteins as indicated . Cells were then subjected to transwell invasion ( F ) assays . The shown images are representative of three independent experiments . Data shown are mean ± SD ( n = 3 ) . * , P ˂ 0 . 05 ; * * * , P ˂ 0 . 001 ; One - way ANOVA . Scale bar : 100 μm .

A549 cells expressing either control shRNA or p53 shRNA were infected with lentiviruses expressing Flag-tagged full-length or truncated WDR63 proteins as indicated. Cells were then subjected to transwell invasion (F) assays. The shown images are representative of three independent experiments. Data shown are mean ± SD (n=3). *, P ˂ 0.05; ***, P ˂ 0.001; One-way ANOVA. Scale bar: 100 μm.


( G ) A549 cells expressing control shRNA , p53 shRNA , or p53 shRNA plus Flag - WDR63 ( each also expressing luciferase ) were injected via tail vein into nude mice ( n = 6 for each group ) . Three weeks after injection , tumor formation was monitored by bioluminescence imaging . Data shown are mean ± SD ; * , P ˂ 0 . 05 ; One - way ANOVA .

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(G) A549 cells expressing control shRNA, p53 shRNA, or p53 shRNA plus Flag-WDR63 (each also expressing luciferase) were injected via tail vein into nude mice (n=6 for each group). Three weeks after injection, tumor formation was monitored by bioluminescence imaging. Data shown are mean ± SD; *, P ˂ 0.05; One-way ANOVA.


Immunoblot ( IB ) analyses of the levels of β - catenin and RAS in DLD1 , WT - HCT116 , MT - HCT116 , HepG2 , and SW48 cells transfected with Myc - axin for 36

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Immunoblot (IB) analyses of the levels of β-catenin and RAS in DLD1, WT-HCT116, MT-HCT116, HepG2, and SW48 cells transfected with Myc-axin for 36 hr


Effects of β - catenin knock - down on polyubiquitination - dependent degradation of RAS . IB analyses of MT - HCT116 cells transfected with control or two independent β - catenin siRNAs for 48

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Effects of β-catenin knock-down on polyubiquitination-dependent degradation of RAS. IB analyses of MT-HCT116 cells transfected with control or two independent β-catenin siRNAs for 48 hr


Polyubiquitination assays of MT - HCT116 cells cotransfected with two independent β - catenin siRNAs and HA - ubiquitin ( Ub ) for 72 hr in the presence of the proteasomal inhibitor MG132 , and WCLs were immunoprecipitated with an anti - pan - RAS

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Polyubiquitination assays of MT-HCT116 cells cotransfected with two independent β-catenin siRNAs and HA-ubiquitin (Ub) for 72 hr in the presence of the proteasomal inhibitor MG132, and WCLs were immunoprecipitated with an anti-pan-RAS antibody


IB analyses of HEK293 cells transfected with WT - or S33Y - Flag - β - catenin with or without HA - GSK3β for 36

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IB analyses of HEK293 cells transfected with WT- or S33Y-Flag-β-catenin with or without HA-GSK3β for 36 hr


IB analyses of HEK293 cells transfected with β - catenin siRNA or control siRNA for 48 h and then treated with L - cell - conditioned medium ( L - CM ) or Wnt3a - CM for 8

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IB analyses of HEK293 cells transfected with β-catenin siRNA or control siRNA for 48 h and then treated with L-cell-conditioned medium (L-CM) or Wnt3a-CM for 8 hr


Effects of KYA1797K or XAV939 on degradation of β - catenin and RAS in isogenic HCT116 cells harboring WT - or MT - CTNNB1 . IB analyses of β - catenin and RAS levels in WT - or MT - HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24

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Effects of KYA1797K or XAV939 on degradation of β-catenin and RAS in isogenic HCT116 cells harboring WT- or MT-CTNNB1. IB analyses of β-catenin and RAS levels in WT- or MT-HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hr


ICC analyses of WT - or MT - HCT116 cells treated with 20 µM of KYA1797K for 24 hr . Scale bars , 10

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ICC analyses of WT- or MT-HCT116 cells treated with 20 µM of KYA1797K for 24 hr. Scale bars, 10 µm


For polyubiquitination assays , WT - or MT - HCT116 cells were transfected with FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132 . WCLs were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB analyses to detect each

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For polyubiquitination assays, WT- or MT-HCT116 cells were transfected with FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132. WCLs were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses to detect each protein


The 3 - ( 4 , 5 - dimethylthiazol - 2yl ) - 2 - 5 - diphenyltetrazolium bromide ( MTT ) assays were performed for measurements of growth of WT - or MT - HCT116 cells treated with the indicated concentrations of KYA1797K . Data are presented as the mean ± SD ( n = 3 )

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The 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assays were performed for measurements of growth of WT- or MT-HCT116 cells treated with the indicated concentrations of KYA1797K. Data are presented as the mean ± SD (n = 3)


Analyses of the interaction between β - catenin and RAS . WCLs of DLD1 cells were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB

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Analyses of the interaction between β-catenin and RAS. WCLs of DLD1 cells were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses


IB analyses of indicated proteins in the samples immunoprecipitated with anti - β - catenin antibody with WCLs of HEK293 cells treated with Wnt3a -

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IB analyses of indicated proteins in the samples immunoprecipitated with anti-β-catenin antibody with WCLs of HEK293 cells treated with Wnt3a-CM


Left panel : MT - HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses , respectively . Right panel : The cytosolic and nuclear fractions were immunoprecipitated with anti - β - catenin antibody and subjected to IB

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Left panel: MT-HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses, respectively. Right panel: The cytosolic and nuclear fractions were immunoprecipitated with anti-β-catenin antibody and subjected to IB analyses


β - Catenin - RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L - CM or Wnt3a - CM . Scale bars , 20 µm . The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100

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β-Catenin-RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L-CM or Wnt3a-CM. Scale bars, 20 µm. The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100 cells


In vitro pulldown analyses to identify β - catenin binding region of RAS . Upper panel : Schematic of the HRAS full - length ( FL ) or two deletion mutants . Lower panel : Purified GST - HRAS - FL , - ΔHVR , or - Δ131 - 165 proteins were pulled - down with purified His - β - catenin - FL protein and subjected to IB

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In vitro pulldown analyses to identify β-catenin binding region of RAS. Upper panel: Schematic of the HRAS full-length (FL) or two deletion mutants. Lower panel: Purified GST-HRAS-FL, -ΔHVR, or -Δ131-165 proteins were pulled-down with purified His-β-catenin-FL protein and subjected to IB analyses


IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - FL , - ΔHVR , or - Δ131 - 165 for 32

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IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-FL, -ΔHVR, or -Δ131-165 for 32 hr


IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - WT , - 2A , or - 2E for 32

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IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-WT, -2A, or -2E for 32 hr


Upper panel : Schematic of the β - catenin - FL or two deletion mutants . Lower panel : Purified His - β - catenin - FL , ΔN86 , or ΔC110 proteins were pull - down with purified GST - HRAS - FL protein and subjected to IB

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Upper panel: Schematic of the β-catenin-FL or two deletion mutants. Lower panel: Purified His-β-catenin-FL, ΔN86, or ΔC110 proteins were pull-down with purified GST-HRAS-FL protein and subjected to IB analyses


IB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc - HRAS and GFP - β - catenin - FL , - ΔN86 , or - ΔC110 for 32

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IB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc-HRAS and GFP-β-catenin-FL, -ΔN86, or -ΔC110 for 32 hr


Fluorescence spectroscopy analyses of the interaction between β - catenin and KRAS using purified recombinant proteins , intact β - catenin and KRAS . Molar ratios of β - catenin and KRAS are presented . A . U . , absorbance

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Fluorescence spectroscopy analyses of the interaction between β-catenin and KRAS using purified recombinant proteins, intact β-catenin and KRAS. Molar ratios of β-catenin and KRAS are presented. A.U., absorbance units


Nuclear magnetic resonance ( NMR ) titration for 15N - labeled KRAS ( full - length , 1 - 188 ) with β - catenin ( C - terminal fragment , 661 - 781 ) . The titration points are indicated in red ( 1 : 0 ) , green ( 1 : 5 ) , and blue ( 1 : 10 ) . A gradual perturbation in chemical shift is indicated by the

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Nuclear magnetic resonance (NMR) titration for 15N-labeled KRAS (full-length, 1-188) with β-catenin (C-terminal fragment, 661-781). The titration points are indicated in red (1:0), green (1:5), and blue (1:10). A gradual perturbation in chemical shift is indicated by the arrow


Effects of KYA1797K on the interaction and phosphorylation of β - catenin and pan - RAS . WT - or MT - HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132 . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB

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Effects of KYA1797K on the interaction and phosphorylation of β-catenin and pan-RAS. WT- or MT-HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses


Effects of Wnt3a - CM treatment on the formation of β - catenin destruction complex and phosphorylation of β - catenin and RAS in HEK293 cells treated with L - CM or Wnt3a - CM in the presence of MG132 for 8 hr . WCLs were immunoprecipitated with β - catenin or pan - RAS antibodies and subjected to IB

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Effects of Wnt3a-CM treatment on the formation of β-catenin destruction complex and phosphorylation of β-catenin and RAS in HEK293 cells treated with L-CM or Wnt3a-CM in the presence of MG132 for 8 hr. WCLs were immunoprecipitated with β-catenin or pan-RAS antibodies and subjected to IB analyses


IB analyses were performed to detect β - catenin and pan - RAS in DLD1 cells cotransfected with FLAG - β - catenin - WT or - MT and Myc - KRAS - WT or - 2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA - GSK3β for 24 hr ,

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IB analyses were performed to detect β-catenin and pan-RAS in DLD1 cells cotransfected with FLAG-β-catenin-WT or -MT and Myc-KRAS-WT or -2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA-GSK3β for 24 hr, respectively


IB analyses to determine the interaction between β - catenin and BBR peptide by in vitro pull - down assays using purified His - β - catenin and GST - BBR

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IB analyses to determine the interaction between β-catenin and BBR peptide by in vitro pull-down assays using purified His-β-catenin and GST-BBR proteins


IB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG - β - catenin and GFP - BBR or - Mock in the presence of proteasome inhibitor , ALLN , for 24

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IB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG-β-catenin and GFP-BBR or -Mock in the presence of proteasome inhibitor, ALLN, for 24 hr


Effects of BBR overexpression on KYA1797K - induced RAS degradation . I analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24

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Effects of BBR overexpression on KYA1797K-induced RAS degradation. I analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr


Effects of BBR overexpression on KYA1797K - induced RAS degradation ICC ( D ; Scale bars , 10 µm ) analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24

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Effects of BBR overexpression on KYA1797K-induced RAS degradation ICC (D; Scale bars, 10 µm) analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr


MT - HCT116 cells were cotransfected with GFP - Mock or - BBR and FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr , and WCLs were immunoprecipitated with pan - RAS

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MT-HCT116 cells were cotransfected with GFP-Mock or -BBR and FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr, and WCLs were immunoprecipitated with pan-RAS antibody


IB analyses of IP with β - catenin antibody in MT - HCT116 cells transfected with GFP - BBR - WT , - 2E , or GFP - Mock in the presence of ALLN for 24

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IB analyses of IP with β-catenin antibody in MT-HCT116 cells transfected with GFP-BBR-WT, -2E, or GFP-Mock in the presence of ALLN for 24 hr


IB analyses of MT - HCT116 cells transfected with GFP - Mock , - BBR - WT , or - BBR - 2E for 24 hr and then treated with 20 µM of KYA1797K for 24

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IB analyses of MT-HCT116 cells transfected with GFP-Mock, -BBR-WT, or -BBR-2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hr


MT - HCT116 cells were transfected with GFP - Mock , - BBR - WT , or - BBR - 2E together with FLAG - Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB

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MT-HCT116 cells were transfected with GFP-Mock, -BBR-WT, or -BBR-2E together with FLAG-Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses


Effects of KYA1797K and PTD - BBR on RAS degradation IB assay of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr ,

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Effects of KYA1797K and PTD-BBR on RAS degradation IB assay of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively


Effects of KYA1797K and PTD - BBR o cell growth MTT assays of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr , respectively . Data are presented as the mean ± SD ( n = 3 )

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Effects of KYA1797K and PTD-BBR o cell growth MTT assays of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively. Data are presented as the mean ± SD (n = 3)


Effects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell

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Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growth


Effects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular

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Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformation


Effects of cotreatments with KYA1797K and PTD - BBR on xenografted tumor growth . MT - HCT116 cells were subcutaneously injected in nude mice with subsequent i . p . injection of vehicle , 20 mg / kg of KYA1797K , or a cotreatment of PTD - BBR ( 25 mg / kg ) and KYA1797K ( 20 mg / kg ) for 20 d . Tumor volumes ( upper panel ) were measured every 4 days , and tumor weights ( lower panel ) were measured at the times of sacrifice . Data are presented as the mean ± SD ( four mice per group )

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

Effects of cotreatments with KYA1797K and PTD-BBR on xenografted tumor growth. MT-HCT116 cells were subcutaneously injected in nude mice with subsequent i.p. injection of vehicle, 20 mg/kg of KYA1797K, or a cotreatment of PTD-BBR (25 mg/kg) and KYA1797K (20 mg/kg) for 20 d. Tumor volumes (upper panel) were measured every 4 days, and tumor weights (lower panel) were measured at the times of sacrifice. Data are presented as the mean ± SD (four mice per group)


WCLs prepared from tumor tissues were immunoprecipitated with anti - β - catenin antibody . WCLs or co - IP samples were subjected to IB

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

WCLs prepared from tumor tissues were immunoprecipitated with anti-β-catenin antibody. WCLs or co-IP samples were subjected to IB analyses


IHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI . Scale bar , 20

IHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI. Scale bar, 20 µm


A Transfected GFP − Atg8 homologs were immunoprecipitated with μMACS ™ from total U2OS cell extracts followed by immunoblot analysis with anti − GFP and anti − ALFY antibodies . Data are representative of two independent experiments .

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A Transfected GFP−Atg8 homologs were immunoprecipitated with μMACS™ from total U2OS cell extracts followed by immunoblot analysis with anti−GFP and anti−ALFY antibodies. Data are representative of two independent experiments.


C The indicated 35S − labelled in vitro − translated GFP − tagged constructs were incubated with GST , GST − LC3B or GST − GABARAP conjugated to glutathione Sepharose , and their binding was evaluated by autoradiography ( ARG ) . 5 % of the in vitro − translated protein ( arrow head ) used was loaded . Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue ( CBB ) staining . Data are representative of three independent experiments .

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

C The indicated 35S−labelled in vitro−translated GFP−tagged constructs were incubated with GST, GST−LC3B or GST−GABARAP conjugated to glutathione Sepharose, and their binding was evaluated by autoradiography (ARG). 5% of the in vitro−translated protein (arrow head) used was loaded. Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments.


D Recombinant ALFY ( aa 2981 - 3526 ) was incubated with GST ‐ Atg8 proteins conjugated to glutathione Sepharose . The pulled ‐ down complexes were subjected to SDS - PAGE and anti ‐ ALFY and anti ‐ GST immunoblotting . 5 % of the recombinant ALFY protein used was loaded as input . Data are representative of three independent experiments .

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D Recombinant ALFY (aa 2981-3526) was incubated with GST‐Atg8 proteins conjugated to glutathione Sepharose. The pulled‐down complexes were subjected to SDS-PAGE and anti‐ALFY and anti‐GST immunoblotting. 5% of the recombinant ALFY protein used was loaded as input. Data are representative of three independent experiments.


B 35S − labelled in vitro − translated GFP − ALFY ( aa 2981 - 3526 ) wild − type and different LIR mutants were incubated with GST − GABARAP or − LC3C and binding evaluated by ARG . 10 and 2 % of the in vitro − translated proteins used were loaded to illustrate binding affinity . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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B 35S−labelled in vitro−translated GFP−ALFY (aa 2981-3526) wild−type and different LIR mutants were incubated with GST−GABARAP or −LC3C and binding evaluated by ARG. 10 and 2% of the in vitro−translated proteins used were loaded to illustrate binding affinity. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


C MBP , MBP − ALFY ( aa 3255 - 3526 ) or the ALFY − LIR mutant ( F3346A ) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP . The pulled − down complexes were subjected to SDS - PAGE and visualized by CBB staining . Data are representative of three independent experiments .

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C MBP, MBP−ALFY (aa 3255-3526) or the ALFY−LIR mutant (F3346A) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP. The pulled−down complexes were subjected to SDS-PAGE and visualized by CBB staining. Data are representative of three independent experiments.


D Thermodynamic parameters of ALFY − LIR peptide ( aa 3341 - 3354 ) to Atg8 homologs . All titrations were performed at 25 ° C as described in Supplementary Methods . ITC data were fitted to a one − site binding model . ND , not detected .

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D Thermodynamic parameters of ALFY−LIR peptide (aa 3341-3354) to Atg8 homologs. All titrations were performed at 25°C as described in Supplementary Methods. ITC data were fitted to a one−site binding model. ND, not detected.


E 35S − labelled in vitro − translated GFP or GFP − Bchs ( aa 3326 - 3341 ) were incubated with GST or GST − dAtg8a and binding evaluated by ARG . 5 % of the in vitro − translated protein used was loaded . Ponceau staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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E 35S−labelled in vitro−translated GFP or GFP−Bchs (aa 3326-3341) were incubated with GST or GST−dAtg8a and binding evaluated by ARG. 5% of the in vitro−translated protein used was loaded. Ponceau staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


D GFP − LC3B and GFP − LC3B ( Q26K / H27Y / H57D ) were immunoprecipitated with GFP − TRAP ® from total cell lysate of stably transfected HeLa FlpIn cells , followed by SDS - PAGE and immunoblotting with the indicated antibodies . Data are representative of three independent experiments .

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

D GFP−LC3B and GFP−LC3B (Q26K/H27Y/H57D) were immunoprecipitated with GFP−TRAP® from total cell lysate of stably transfected HeLa FlpIn cells, followed by SDS-PAGE and immunoblotting with the indicated antibodies. Data are representative of three independent experiments.


E MBP ‐ ALFY3255 - 3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants . The pulled ‐ down complexes were subjected to SDS - PAGE and visualized by immunoblotting with anti ‐ MBP and anti ‐ GABARAP antibodies . Data are representative of three independent experiments .

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E MBP‐ALFY3255-3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants. The pulled‐down complexes were subjected to SDS-PAGE and visualized by immunoblotting with anti‐MBP and anti‐GABARAP antibodies. Data are representative of three independent experiments.


F MBP − tagged ALFY3255 - 3526 and p62168 - 391 or their corresponding LIR mutants ( F3346A and W340A , respectively ) were conjugated to amylose resin and incubated with purified GST , GABARAP , GABARAP mutant ( K24Q / Y25H / D54H ) , LC3B or LC3B mutant ( Q26K / H27Y / H57D ) . The bound proteins were visualized by immunoblotting with anti − GABARAP , anti − LC3 and anti − MBP antibodies . Data are representative of three independent experiments .

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F MBP−tagged ALFY3255-3526 and p62168-391 or their corresponding LIR mutants (F3346A and W340A, respectively) were conjugated to amylose resin and incubated with purified GST, GABARAP, GABARAP mutant (K24Q/Y25H/D54H), LC3B or LC3B mutant (Q26K/H27Y/H57D). The bound proteins were visualized by immunoblotting with anti−GABARAP, anti−LC3 and anti−MBP antibodies. Data are representative of three independent experiments.


G 35S ‐ labelled in vitro ‐ translated GFP ‐ ALFY ( aa 2981 - 3526 ) wild ‐ type and K3343A / D3344A / Y3351A mutant were incubated with GST ‐ LC3B and GABARAP and binding evaluated by ARG . 10 % of the in vitro ‐ translated proteins used was loaded . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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

G 35S‐labelled in vitro‐translated GFP‐ALFY (aa 2981-3526) wild‐type and K3343A/D3344A/Y3351A mutant were incubated with GST‐LC3B and GABARAP and binding evaluated by ARG. 10% of the in vitro‐translated proteins used was loaded. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


A HeLa FlpIn GFP − GABARAP cells were treated with proteasomal inhibitor ( MG132 , 2 h ) or subjected to amino acid starvation ( EBSS , 2 h ) , before staining with anti − ALFY antibodies . Scale bar , 10 μm .

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A HeLa FlpIn GFP−GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti−ALFY antibodies. Scale bar, 10 μm.


B GFP − tagged full − length ALFY wild − type or LC3 − interacting region ( LIR ) − mutant was expressed into Alfy − deficient MEFs using an adenovirus system . At 48 h after infection , the MEFs were cultured in normal media or EBSS for 1 . 5 h before staining with anti − LC3B or anti − GABARAP antibodies . Scale bars 10 μm .

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B GFP−tagged full−length ALFY wild−type or LC3−interacting region (LIR)−mutant was expressed into Alfy−deficient MEFs using an adenovirus system. At 48 h after infection, the MEFs were cultured in normal media or EBSS for 1.5 h before staining with anti−LC3B or anti−GABARAP antibodies. Scale bars 10 μm.


C HeLa cells were treated with control or siRNA targeting GABARAP , GABARAPL1 and GABARAPL2 . 72 h after transfection cells were immunostained with anti − LC3B , anti − ALFY and anti − p62 antibodies . Scale bars , 10 μm .

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C HeLa cells were treated with control or siRNA targeting GABARAP, GABARAPL1 and GABARAPL2. 72 h after transfection cells were immunostained with anti−LC3B, anti−ALFY and anti−p62 antibodies. Scale bars, 10 μm.


D HeLa cells were treated with control or siRNA targeting ALFY . 72 h after transfection , cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX − 100 − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments .

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D HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX−100−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.


E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X − 100 ( TX − 100 ) − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments .

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E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X−100 (TX−100)−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.


Ala / Phe substitutions of aa 239 - 244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity . Aβ38 , Aβ40 , and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA . Aβ ( 38 + 40 ) / 42 ratio was calculated to determine GSEC processivity towards APPC99 . DKO PSEN1 / PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references .

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

Ala/Phe substitutions of aa 239-244 were introduced in the NCT ectodomain to evaluate the role of this region in the regulation of GSEC processivity. Aβ38, Aβ40, and Aβ42 levels present in the conditioned medium collected from KO NCT MEF cells rescued with WT or respective mutant NCT GSECs and transiently expressing with APPC99 were quantified by ELISA. Aβ(38+40)/42 ratio was calculated to determine GSEC processivity towards APPC99. DKO PSEN1/PSEN2 MEFs rescued with the indicated FAD PSEN1 mutant and transduced with APPC99 were used as references.


Representative SDS - PAGE / western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits . The presence of mature , glycosylated NCT , N - terminal and C - terminal fragments of the endoproteolysed PSEN1 and PEN - 2 , compared to NCT knock - out ( KO ) cells , indicate that WT and mutated NCTs reconstitute GSEC complexes . Arrowheads indicate the position of molecular weight markers .

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Representative SDS-PAGE/western blot analysis of CHAPSO solubilized membranes from KO NCT MEF cell lines stably expressing WT or mutant NCT subunits. The presence of mature, glycosylated NCT, N-terminal and C-terminal fragments of the endoproteolysed PSEN1 and PEN-2, compared to NCT knock-out (KO) cells, indicate that WT and mutated NCTs reconstitute GSEC complexes. Arrowheads indicate the position of molecular weight markers.


Quantification of the effects of the indicated substitutions of aa 241 , 242 , and 244 in NCT on GSEC processivity of APPC99 , estimated as indicated above by the Aβ ( 38 + 40 ) / 42 ratio . Figure EV1B shows the corresponding Aβ42 / 40 ratios .

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Quantification of the effects of the indicated substitutions of aa 241, 242, and 244 in NCT on GSEC processivity of APPC99, estimated as indicated above by the Aβ(38+40)/42 ratio. Figure EV1B shows the corresponding Aβ42/40 ratios.


( Top ) Representative bright field microscopy images show MEF cells stably expressing WT or NCT mutant GSECs after 24h incubation at 37 ° C and 42 ° C . 25µm scale bar is shown . ( Bottom ) Cell viability determined at the indicated time points is not affected by prolonged ( ≥ 24h ) incubation at 42 ° C . Data are presented as mean ± SD of 4 replicates .

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(Top) Representative bright field microscopy images show MEF cells stably expressing WT or NCT mutant GSECs after 24h incubation at 37°C and 42°C. 25µm scale bar is shown. (Bottom) Cell viability determined at the indicated time points is not affected by prolonged (≥ 24h) incubation at 42°C. Data are presented as mean ± SD of 4 replicates.


Relative GSEC - APP / Aβn stabilities were assessed in cell - based GSEC thermoactivity assays , which evaluate changes in GSEC processivity ( Aβ ( 38 + 40 ) / 42 ratio ) upon incubation at 42 ° C for 24h , relative to 37 ° C . Aβ peptides in the conditioned medium collected from the indicated MEF cell lines ( transiently expressing APPC99 ) were quantified and the Aβ ( 38 + 40 ) / 42 ratios determined . The data are shown as mean ± 95 % CI , as % of the WT GSEC cell line incubated at 37 ° C for 24h , N = 6 independent experiments . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine the statistical significance ( F ( DFn , DFd ) : F ( 6 , 46 ) = 100 ) ; * * * * p > 0 . 0001 compared to WT at 37 ° C , # p > 0 . 05 , # # p > 0 . 01 , # # # # p > 0 . 0001 compared to WT at 42 ° C . Note : Aβ profiles ( including Aβ43 levels ) normalized to total Aβ quantified in the cell - based thermoactivity assays with the different mNCT WT / mutants are shown in Figure EV2 .

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

Relative GSEC-APP/Aβn stabilities were assessed in cell-based GSEC thermoactivity assays, which evaluate changes in GSEC processivity (Aβ(38+40)/42 ratio) upon incubation at 42°C for 24h, relative to 37°C. Aβ peptides in the conditioned medium collected from the indicated MEF cell lines (transiently expressing APPC99) were quantified and the Aβ(38+40)/42 ratios determined. The data are shown as mean ± 95% CI, as % of the WT GSEC cell line incubated at 37°C for 24h, N = 6 independent experiments. One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine the statistical significance (F(DFn, DFd): F(6, 46) = 100); ****p > 0.0001 compared to WT at 37°C, #p > 0.05, ##p>0.01, ####p>0.0001 compared to WT at 42°C. Note: Aβ profiles (including Aβ43 levels) normalized to total Aβ quantified in the cell-based thermoactivity assays with the different mNCT WT/mutants are shown in Figure EV2.


Aβ profiles ( % contribution of individual Aβ37 , Aβ38 , Aβ40 , and Aβ42 peptides to the total Aβ levels ( Aβ37 + 38 + 40 + 42 ) ) in the conditioned medium collected from WT or mutant ( NCT ) MEF cells lines co - expressing WT or mutant APPC99 substrates were assayed by ELISA . Schematic models of the tested GSEC - APP complexes are shown .

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Aβ profiles (% contribution of individual Aβ37, Aβ38, Aβ40, and Aβ42 peptides to the total Aβ levels (Aβ37+38+40+42)) in the conditioned medium collected from WT or mutant (NCT) MEF cells lines co-expressing WT or mutant APPC99 substrates were assayed by ELISA. Schematic models of the tested GSEC-APP complexes are shown.


To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions , the ratio of ( ( Aβ 37 / 40 ) + ( Aβ 38 / 42 ) ) / 2 was determined and used to estimate GSEC processivity of APPC99 . All the data are presented as mean ± SD , N ≥ 3 , Unpaired , two - tailed Student ' s t - test was used to determine the statistical significance . * * p > 0 . 01 , * * * * p > 0 . 0001 . Calculated T - and Df values were : T = 11 , 63 ; Df = 16 ( I241 - K28 vs I241E - K28 ) , T = 11 , 37 ; Df = 14 ( I241 - K28 vs I241 - K28E ) , T = 4 , 572 ; Df = 8 ( I241E - K28 vs I241 - K28E ) , T = 4 , 497 ; Df = 5 ( I241 - K28E vs I241K - K28E ) , T = 2 , 561 ; Df = 7 .

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To account for the increased production of Aβ37 and Aβ38 at the expense of Aβ40 and Aβ42 in the tested conditions, the ratio of ((Aβ 37/40) + (Aβ 38/42))/2 was determined and used to estimate GSEC processivity of APPC99. All the data are presented as mean ± SD, N ≥ 3, Unpaired, two-tailed Student's t-test was used to determine the statistical significance. **p > 0.01, ****p > 0.0001. Calculated T- and Df values were: T = 11,63; Df = 16 (I241-K28 vs I241E-K28), T = 11,37; Df = 14 (I241-K28 vs I241-K28E), T = 4,572; Df = 8 (I241E-K28 vs I241-K28E), T = 4,497; Df = 5 (I241-K28E vs I241K-K28E), T = 2,561; Df = 7.