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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

(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

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

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

O O O O O O O O O O O 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 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

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

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

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

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

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

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

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

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

O O O 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

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 )

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

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

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 -

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ,

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

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

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

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

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

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

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

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

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 B-CONTROLLED_VAR O O

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

O O O 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

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

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

O O O O O O O O O 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

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

O O O O O O O O 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

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 )

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

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

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

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

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 )

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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

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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 .

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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 .

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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 .

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 .

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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 .

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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 .

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

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 .

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

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 .

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

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.


Introduction of Cys at position 241 in NCT allows chemical cross - linking of NCT and APPC99 with BMPS . Representative SDS - PAGE / western blot show cross - linked NCT - APPC99 , NCT and APPC99 bands in detergent - extracted membrane proteins from BMPS cross - linker - treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate . Orange , black , and white arrows indicate the NCT - APPC99 / Aβ crosslinked , mature NCT , and immature NCT bands , respectively . Arrowheads indicate the position of molecular weight markers . Analysis of the western blot density profiles in panel ( B ) shows that co - migration of NCT ( orange arrow ) with APPC99 shifts the cross - linked band towards a higher molecular weight than the observed for mature NCT ( black arrow ) . Immature NCT is indicated with an open ( white ) arrow . Full - range molecular weight density profiles are shown in Figure

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

Introduction of Cys at position 241 in NCT allows chemical cross-linking of NCT and APPC99 with BMPS. Representative SDS-PAGE/western blot show cross-linked NCT-APPC99, NCT and APPC99 bands in detergent-extracted membrane proteins from BMPS cross-linker-treated membranes prepared from MEF cell lines expressing the indicated WT or mutant NCT GSEC complexes together with APPC99 or in the absence of the substrate. Orange, black, and white arrows indicate the NCT-APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the western blot density profiles in panel (B) shows that co-migration of NCT (orange arrow) with APPC99 shifts the cross-linked band towards a higher molecular weight than the observed for mature NCT (black arrow). Immature NCT is indicated with an open (white) arrow. Full-range molecular weight density profiles are shown in Figure EV3C


Representative SDS - PAGE / western blot presents NCT - APPC99 , NCT and APPC99 expression in detergent - extracted membranes prepared from MEF cell lines co - expressing the mutant NCT - I241C GSEC complex with the APPC99 - K28C substrate . As negative control , the same sample was supplemented with β - mercaptoethanol ( BME ) to reduce all disulfide bridges . A full - range molecular weight western blot is shown in Figure EV3B . Orange , black , and white arrows indicate the NCT - APPC99 / Aβ crosslinked , mature NCT , and immature NCT bands , respectively . Arrowheads indicate the position of molecular weight markers . Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT - I241C andAPPC99 K28C ( mature and immature NCT are indicated with black and white arrows , respectively . The filled orange arrow points at the high molecular weight APPC99 / Aβ density ( crosslinked NCT - APPC99 / Aβ band ) . Note that peak amplitudes for the cross - linked NCT - APPC99 and the mature NCT band were normalized to similar values for clarity purposes . Full - range molecular weight density profiles are shown in Figure EV3D .

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

Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT -APPC99/Aβ crosslinked, mature NCT, and immature NCT bands, respectively. Arrowheads indicate the position of molecular weight markers. Analysis of the integrated density profiles of the respective western blot bands supports the spontaneous formation of a disulfide bond between NCT- I241C andAPPC99 K28C (mature and immature NCT are indicated with black and white arrows, respectively. The filled orange arrow points at the high molecular weight APPC99/Aβ density (crosslinked NCT-APPC99/Aβ band). Note that peak amplitudes for the cross-linked NCT-APPC99 and the mature NCT band were normalized to similar values for clarity purposes. Full-range molecular weight density profiles are shown in Figure EV3D.


Representative SDS - PAGE / western blot presents NCT - APPC99 , NCT and APPC99 expression in detergent - extracted membranes prepared from MEF cell lines co - expressing the mutant NCT - I241C GSEC complex with the APPC99 - K28C substrate . As negative control , the same sample was supplemented with β - mercaptoethanol ( BME ) to reduce all disulfide bridges . A full - range molecular weight western blot is shown in Figure EV3B . Orange , black , and white arrows indicate the

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

Representative SDS-PAGE/western blot presents NCT-APPC99, NCT and APPC99 expression in detergent-extracted membranes prepared from MEF cell lines co-expressing the mutant NCT- I241C GSEC complex with the APPC99-K28C substrate. As negative control, the same sample was supplemented with β-mercaptoethanol (BME) to reduce all disulfide bridges. A full-range molecular weight western blot is shown in Figure EV3B. Orange, black, and white arrows indicate the NCT


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 vehicle ( DMSO ) or GSM - treated ( 1 µM final concentration ) MEF cells co - expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA . Data generated using 0 . 3 µM GSM - treated MEF cells is shown in Figure EV4 . Data are presented as mean ± SD , N ≥ 3 independent experiments .

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

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 vehicle (DMSO) or GSM-treated (1 µM final concentration) MEF cells co-expressing respective WT or mutant NCT GSEC with APPC99 were determined by ELISA. Data generated using 0.3 µM GSM-treated MEF cells is shown in Figure EV4. Data are presented as mean ± SD, N ≥ 3 independent experiments.


Aβ 37 / 40 and Aβ 38 / 42 ratios were calculated to evaluate the efficiency of the GSEC - mediated cleavage of APPC99 ( Figure EV4 ) and statistical analysis ( P values ) ( two - way ANOVA with Sidak ' s post hoc test ) for the effect of GSMs on WT and mutant complexes is presented ( response to GSM normalized to the corresponding vehicle condition ) .

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

Aβ 37/40 and Aβ 38/42 ratios were calculated to evaluate the efficiency of the GSEC-mediated cleavage of APPC99 (Figure EV4) and statistical analysis (P values) (two-way ANOVA with Sidak's post hoc test) for the effect of GSMs on WT and mutant complexes is presented (response to GSM normalized to the corresponding vehicle condition).


A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1 / S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points .

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A Immunoblot analysis of HeLa cells using the indicated antibodies that were synchronized in G1/S phase using a double thymidine block or in mitosis using sequential thymidine and nocodazole treatment and collected at the given time points.


B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment .

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B Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides as indicated and left untreated or synchronized in mitosis using sequential thymidine and nocodazole treatment.


C Co - immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG - tagged XIAP or empty vector ( EV ) and synchronized in mitosis using nocodazole or left untreated .

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C Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were transfected with FLAG-tagged XIAP or empty vector (EV) and synchronized in mitosis using nocodazole or left untreated.


D Co - immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in ( C ) , but with additional transfection of the FLAG - tagged XIAP single amino acid mutants G188R and G188E . Only mitotic samples are shown . The asterisk denotes ubiquitylated forms of XIAP .

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D Co-immunoprecipitation of XIAP and endogenous USP9X in HEK 293T cells that were treated as in (C), but with additional transfection of the FLAG-tagged XIAP single amino acid mutants G188R and G188E. Only mitotic samples are shown. The asterisk denotes ubiquitylated forms of XIAP.


E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG - tagged XIAP and transfected with siRNA oligonucleotides as specified . Cells were synchronized in mitosis using sequential thymidine / nocodazole treatment , as indicated . Subsequent to treatment with MG132 , whole cell extracts ( WCE ) were prepared and ubiquitylated XIAP was isolated by anti - FLAGimmunoprecipitation ( IP ) under denaturing conditions .

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E In vivo ubiquitylation of XIAP in HeLa cells that were infected with the indicated expression constructs carrying FLAG-tagged XIAP and transfected with siRNA oligonucleotides as specified. Cells were synchronized in mitosis using sequential thymidine/nocodazole treatment, as indicated. Subsequent to treatment with MG132, whole cell extracts (WCE) were prepared and ubiquitylated XIAP was isolated by anti-FLAGimmunoprecipitation (IP) under denaturing conditions.


F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S . The band in the EV control lane of the anti - V5 panel marks an unspecific band produced by the antibody .

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F Immunoblot analysis of NIH 3T3 cells that were transfected with expression constructs for USP9XWT or the catalytically inactive mutant USP9XC1556S. The band in the EV control lane of the anti-V5 panel marks an unspecific band produced by the antibody.


G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine / nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated .

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G Immunoblot analysis of HeLa cells using antibodies to the indicated endogenous proteins that were synchronized in mitosis using thymidine/nocodazole and treated with DMSO or the USP9X inhibitor WP1130 as indicated.


A Immunoblot analysis of WT , Xiap - / - , Mcl1 - / - or cIap2 - / - mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non - relevant mRNA ( Ctrl ) or against Usp9X mRNA and treated with taxol as specified .

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A Immunoblot analysis of WT, Xiap-/-, Mcl1-/- or cIap2-/- mouse embryonic fibroblasts that were lentivirally infected with shRNA constructs directed against a non-relevant mRNA (Ctrl) or against Usp9X mRNA and treated with taxol as specified.


B Two - dimensional cell - cycle analysis ( BrdU / PI ) of cells described in ( A ) . Sub G1 and G2 / M fractions of cells were quantified and averaged with two additional , independent experiments ( n = 3 , ± SD ; WT : * * , P = 0 . 0016 ; * * * , P = 0 . 0004 ; Mcl1 - / - : * * * , P = 0 . 0003 ; * * , P = 0 . 0017 ; cIap2 - / - : * * , P = 0 . 0043 , Student ' s t test ) . Black bars exemplify shRNA Ctrl , white bars shRNA Usp9X samples .

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B Two-dimensional cell-cycle analysis (BrdU/PI) of cells described in (A). Sub G1 and G2/M fractions of cells were quantified and averaged with two additional, independent experiments (n=3, ± SD; WT: **, P = 0.0016; ***, P = 0.0004; Mcl1-/-: ***, P = 0.0003; **, P = 0.0017; cIap2-/-: **, P = 0.0043, Student's t test). Black bars exemplify shRNA Ctrl, white bars shRNA Usp9X samples.


C Immunoblot analysis of HeLa cells transfected with a FLAG - tagged USP9X expression construct or empty vector ( EV ) and treated with taxol for the indicated times .

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C Immunoblot analysis of HeLa cells transfected with a FLAG-tagged USP9X expression construct or empty vector (EV) and treated with taxol for the indicated times.


D Immunoblot analysis of HeLa cells that were transfected with a FLAG - tagged XIAP expression construct or empty vector and treated as in ( C ) .

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D Immunoblot analysis of HeLa cells that were transfected with a FLAG-tagged XIAP expression construct or empty vector and treated as in (C).


E Two dimensional cell - cycle analysis ( BrdU / PI ) of cells described in ( C ) and ( D ) . Apoptotic indices represent ratios of sub G1 to G1 / S cells and are shown for analyses at the indicated timepoints . * * , P = 0 . 0020 ; * * * , P = 0 . 0004 ; Student ' s t test .

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E Two dimensional cell-cycle analysis (BrdU/PI) of cells described in (C) and (D). Apoptotic indices represent ratios of sub G1 to G1/S cells and are shown for analyses at the indicated timepoints. **, P = 0.0020; ***, P = 0.0004; Student's t test.


F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non - relevant mRNA ( Ctrl ) or against Xiap mRNA and treated with taxol as specified .

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F Immunoblot analysis of HeLa cells that were transfected with siRNA oligonucleotides directed against a non-relevant mRNA (Ctrl) or against Xiap mRNA and treated with taxol as specified.


A Immunoblot analysis of sorted Eμ - Myc lymphoma cells that were retrovirally transduced with the indicated IRES - GFP shRNA constructs .

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A Immunoblot analysis of sorted Eμ-Myc lymphoma cells that were retrovirally transduced with the indicated IRES-GFP shRNA constructs.


B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ - Myc lymphoma cells transduced with the indicated shRNA constructs as described in ( A ) . Prior to injection , cells were sorted for viability ( propidium iodide negative ) and GFP positivity ( sh _ Ctrl , black , n = 6 ; sh _ USP9X , gray , n = 5 ) . * P = 0 . 0293 ; Mantel - Cox test .

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B Kaplan Meier survival curves of mice that were injected intravenously with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A). Prior to injection, cells were sorted for viability (propidium iodide negative) and GFP positivity (sh_Ctrl, black, n = 6; sh_USP9X, gray, n = 5). *P = 0.0293; Mantel-Cox test.


C Kaplan - Meier survival curves of mice that were injected with syngeneic Eμ - Myc lymphoma cells transduced with the indicated shRNA constructs as described in ( A ) and treated as in ( B ) ( sh _ Ctrl , black , n = 6 ; sh _ XIAP , gray , n = 6 ) . * * , P = 0 . 0020 ; Mantel - Cox test .

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C Kaplan-Meier survival curves of mice that were injected with syngeneic Eμ-Myc lymphoma cells transduced with the indicated shRNA constructs as described in (A) and treated as in (B) (sh_Ctrl, black, n = 6; sh_XIAP, gray, n = 6). **, P = 0.0020; Mantel-Cox test.


D Histopathology of representative lymph nodes derived from mice shown in ( B ) to visualize histomorphology by haematoxylin / eosin ( H & amp ; amp ; E ) , B - cell origin ( B220 ) , and spontaneous apoptosis ( cleaved Caspase 3 ) in situ . Scale bars denote 50 μm and 20 μm for insets .

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D Histopathology of representative lymph nodes derived from mice shown in (B) to visualize histomorphology by haematoxylin/eosin (H&amp;E), B-cell origin (B220), and spontaneous apoptosis (cleaved Caspase 3) in situ. Scale bars denote 50 μm and 20 μm for insets.


E Histopathology of representative lymph nodes derived from mice shown in ( C ) which were stained as described in ( D ) .

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E Histopathology of representative lymph nodes derived from mice shown in (C) which were stained as described in (D).


F Mean tumor burden in mice injected with Eμ - Myc lymphoma cells modified as in ( B ) and ( C ) , 15 days after injection , assessed by PET imaging ( 4 animals per group ) . * * , P = 0 . 0025 ; * * * , P = 0 . 0003 , student ' s t -

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F Mean tumor burden in mice injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection, assessed by PET imaging (4 animals per group). **, P = 0.0025; ***, P = 0.0003, student's t-test


G Representative PET images of animals , injected with Eμ - Myc lymphoma cells modified as in ( B ) and ( C ) , 15 days after injection ( pre treatment ) and three days following intraperitoneal vincristine administration ( post treatment ) . Bone marrow infiltration , representative for systemic lymphoma manifestation , is indicated by a blue asterisk .

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G Representative PET images of animals, injected with Eμ-Myc lymphoma cells modified as in (B) and (C), 15 days after injection (pre treatment) and three days following intraperitoneal vincristine administration (post treatment). Bone marrow infiltration, representative for systemic lymphoma manifestation, is indicated by a blue asterisk.


H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden ( sh _ Ctrl , black , n = 6 ; sh _ Usp9X , white , n = 8 ; sh _ Xiap , grey , n = 8 ) . Within the sh _ Usp9X and sh _ Xiap groups all animals lived up to control imaging , while two of eight animals of the sh _ ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden . These animals had to be excluded from final analysis , suggesting an underestimation of the actual effect . Data was obtained from two independent experiments . * * * * , P < 0 . 0001 ; * , P = 0 . 0167 , student ' s t -

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H Mean change in standardized 18FDG uptake of ROI between pre and post treatment imaging corrected for initial tumor burden (sh_Ctrl, black, n=6; sh_Usp9X, white, n=8; sh_Xiap, grey, n=8). Within the sh_Usp9X and sh_Xiap groups all animals lived up to control imaging, while two of eight animals of the sh_ctrl group had to be sacrificed prematurely because they had reached the predefined criteria of maximum tumor burden. These animals had to be excluded from final analysis, suggesting an underestimation of the actual effect. Data was obtained from two independent experiments. ****, P < 0.0001; *, P = 0.0167, student's t-test


A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins .

A Immunoblot analyses of different human DLBCL lines using antibodies to the indicated proteins.


B FACS analysis ( propidium iodide ( PI ) ) of USP9X high and low expressing DLBCL cells shown in ( A ) to determine cell survival . Cells were treated with taxol and collected at the specified time points . PI negative cells are indicated relative to time point 0 ( n = 3 ± SD ) .

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B FACS analysis (propidium iodide (PI)) of USP9X high and low expressing DLBCL cells shown in (A) to determine cell survival. Cells were treated with taxol and collected at the specified time points. PI negative cells are indicated relative to time point 0 (n=3 ± SD).


C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES - GFP shRNA constructs against USP9X or a non - relevant mRNA , FACS sorted for GFP + PI - cells and exposed to taxol for the indicated periods of time . The quantification of cleaved caspase 3 ( CC3 ) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA . * * , P = 0 . 0099 , student ' s t -

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C Immunoblot analyses of the indicated DLBCL cell lines that were lentivirally transduced with IRES-GFP shRNA constructs against USP9X or a non-relevant mRNA, FACS sorted for GFP+ PI- cells and exposed to taxol for the indicated periods of time. The quantification of cleaved caspase 3 (CC3) at the 24 hr post taxol treatment timepoint from four independent experiments is quantified in the graph on the right side and normalized to control shRNA. **, P = 0.0099, student's t-test


D Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time . Three hours before collecting , WP1130 at a concentration of 5μM or DMSO was added as specified .

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D Immunoblot analyses of the indicated DLBCL cell lines that were exposed to taxol for the indicated periods of time. Three hours before collecting, WP1130 at a concentration of 5μM or DMSO was added as specified.


E FACS analysis ( propidium iodide ( PI ) uptake ) of DLBCL cell lines treated with taxol and / or the SMAC mimetic BV6 as indicated . Results displayed are from three independent experiments ( n = 3 ± SD ) . * * * , P = 0 . 00085 ; * * * , P = 0 . 00529 ; Student ' s t test .

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E FACS analysis (propidium iodide (PI) uptake) of DLBCL cell lines treated with taxol and/or the SMAC mimetic BV6 as indicated. Results displayed are from three independent experiments (n=3 ± SD). ***, P = 0.00085; ***, P = 0.00529; Student's t test.


F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays ( TMA ) derived from a patient cohort representing 121 evaluable human CD20 + aggressive B - cell lymphomas . High USP9X expression was identified in 37 cases ( 31 % ) and significantly correlated with high XIAP expression levels ( * * , P = 0 . 004 ; Student ' s t test ) .

F Collective assessment of USP9X and XIAP expression determined by immunohistochemistry in tissue microarrays (TMA) derived from a patient cohort representing 121 evaluable human CD20+ aggressive B-cell lymphomas. High USP9X expression was identified in 37 cases (31 %) and significantly correlated with high XIAP expression levels (**, P = 0.004; Student's t test).


G Expression data derived from ( E ) was correlated with clinical follow - up . Within a cohort of 58 patients receiving vincristine - containing chemotherapy ( CHOP regimen : cyclophosphamide , doxorubicin , vincristine , prednisone ) without the addition of the anti - CD20 antibody rituximab ( left panel ) overexpression of USP9X and XIAP ( n = 14 , straight line ) was associated with a significantly shortened event - free survival ( EFS ) as compared to all remaining patients ( n = 44 , dotted line ) ( * , P = 0 . 050 ; log rank test ) . Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti - CD20 antibody rituximab overexpression of USP9X and XIAP ( n = 14 , straight line ) was not associated with a shortened event - free survival ( EFS ) as compared to all remaining patients ( n = 49 , dotted line ) ( P = 0 . 454 ) .

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G Expression data derived from (E) was correlated with clinical follow-up. Within a cohort of 58 patients receiving vincristine-containing chemotherapy (CHOP regimen: cyclophosphamide, doxorubicin, vincristine, prednisone) without the addition of the anti-CD20 antibody rituximab (left panel) overexpression of USP9X and XIAP (n = 14, straight line) was associated with a significantly shortened event-free survival (EFS) as compared to all remaining patients (n = 44, dotted line) (*, P = 0.050; log rank test). Within a cohort of 63 patients receiving the CHOP regimen with addition of the anti-CD20 antibody rituximab overexpression of USP9X and XIAP (n = 14, straight line) was not associated with a shortened event-free survival (EFS) as compared to all remaining patients (n = 49, dotted line) (P = 0.454).


molecular detection of nCoV - 2019 in seven patients during two times of sampling . Patient information can be found in Extended Data Table 1 and 2 . Details on detection method can be found in material and methods . BALF , bronchoalveolar lavage fluid ; OS , oral swab ; AS , anal swab .

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molecular detection of nCoV-2019 in seven patients during two times of sampling. Patient information can be found in Extended Data Table 1 and 2. Details on detection method can be found in material and methods. BALF, bronchoalveolar lavage fluid; OS, oral swab; AS, anal swab.


dynamics of nCoV - 2019 antibodies in one patient who showed sign of disease on 2019 . 12 . 23 ( ICU - 06 ) . For b and c , cut - off was set up as 0 . 2 for IgM test and 0 . 3 for IgG test , according to healthy controls .

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dynamics of nCoV-2019 antibodies in one patient who showed sign of disease on 2019.12.23 (ICU-06). For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


serological test of nCoV - 2019 antibodies in five patients ( more information can be found in Extended Data Table 2 ) . Star indicates data collected from patient ICU - 06 on 2020 . 01 . 10 . For b and c , cut - off was set up as 0 . 2 for IgM test and 0 . 3 for IgG test , according to healthy controls .

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serological test of nCoV-2019 antibodies in five patients (more information can be found in Extended Data Table 2). Star indicates data collected from patient ICU-06 on 2020.01.10. For b and c, cut-off was set up as 0.2 for IgM test and 0.3 for IgG test, according to healthy controls.


viral particles in the ultrathin sections under electron microscope at 200 kV , sample from viral infected Vero E6

viral particles in the ultrathin sections under electron microscope at 200 kV, sample from viral infected Vero E6 cells


usage . Determination of virus infectivity in HeLa cells with or without the expression of ACE2 . h , human ; b , Rhinolophus sinicus bat ; c , civet ; s , swine ( pig ) ; m , mouse . ACE2 protein ( green ) , viral protein ( red ) and nuclei ( blue ) was shown . Scale bar = 10 um .

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usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.


A . RT - PCR analysis of the expression of β - actin , Trpv1 , Trpv2 , Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 ( upper ) and 35 ( lower ) thermal cycles with ( RT ( + ) ) and without ( RT ( - ) ) reverse transcription ( RT ) . Control ( Ct . ) lanes indicate the results with each plasmid DNA as a template .

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A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template.


B . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes . Expression levels of mRNA were normalized to that of the ribosomal protein gene ( 36B4 ) , a housekeeping gene un - affected by adipogenesis . Data are presented as mean ± SEM , n = 6 .

B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6.


C . Results of real - time PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue ( iBAT ) . mRNA expression levels were normalized to that of 36B4 . Data are presented as mean ± SEM , n = 5 .

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C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5.


D . Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes . Upper bands in the TRPV2 blots likely indicate glycosylated forms .

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D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms.


E . Averaged traces of [ Ca2 + ] i changes in response to 500 µmol / L 2APB in differentiated brown adipocytes from WT ( black ) and TRPV2KO ( red ) mice . One µmol / L NE was used to confirm differentiation . Five µmol / L ionomycin was used to confirm cell viability . Ratio values correspond to the real [ Ca2 + ] i of differentiated mousebrown adipocytes . Data are presented as mean ± SEM , n = 149 of WT cells , and n = 112 of TRPV2KO brown adipocytes ; * * P < 0 . 01 . Unpaired Student ' s t - test .

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E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test.


F . A representative trace of whole - cell current activated by 3 mmol / L of 2APB in the presence or absence of 10 µmol / L SKF in mouse differentiated brown adipocyte . The left inset indicates a voltage ramp - pulse protocol . The right inset indicates the current - voltage curves of basal current ( a , black ) , current in the presence of 2APB + SKF ( b , blue ) and current in the presence of 2APB alone ( c , red ) at the time points of a , b and c , respectively . G . Comparison of the mean densities of basal currents ( Base , black ) , currents in the presence of 2APB alone ( red ) and currents in the presence of 2APB + SKF ( blue ) at - 60 mV and + 100 mV in mouse differentiated brown adipocytes . Data are presented as mean ± SEM , n = 10 ; * * P < 0 . 01 vs . Base ; # # P < 0 . 01 vs . 2APB alone . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Results of real - time RT - PCR analysis of Trpv1 , Trpv2 , Trpv3 and Trpv4 expression in mouse pre - adipocytes and differentiated brown adipocytes . Expression levels of mRNA were normalized to those of 36B4 . Data are presented as mean ± SEM , n = 6 . * P < 0 . 05 vs . pre - adipocytes . Unpaired Student ' s t - test .

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A. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression in mouse pre-adipocytes and differentiated brown adipocytes. Expression levels of mRNA were normalized to those of 36B4. Data are presented as mean ± SEM, n = 6. * P < 0.05 vs. pre-adipocytes. Unpaired Student's t-test.


B . The number of 6 day - differentiated mousebrown adipocytes treated with TRPV2 agonists , 100 µmol / L 2APB or 10 µmol / L LPC . Mean ± SEM , n = 6 , * P < 0 . 05 and * * P < 0 . 01 vs . dimethyl sulfoxide ( DMSO ) group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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B. The number of 6 day-differentiated mousebrown adipocytes treated with TRPV2 agonists, 100 µmol/L 2APB or 10 µmol/L LPC. Mean ± SEM, n = 6, * P < 0.05 and ** P < 0.01 vs. dimethyl sulfoxide (DMSO) group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


C and D . Comparison of the numbers of 6 day - differentiated mouse brown adipocytes ( C ) and triglyceride levels ( D ) in the cells from WT and TRPV2KO mice with different differentiation media . 1 / 10 suppl . indicates differentiation medium ten - time - diluted with DMEM . Mean ± SEM , n = 8 , * * P < 0 . 01 vs . control group , # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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C and D. Comparison of the numbers of 6 day-differentiated mouse brown adipocytes (C) and triglyceride levels (D) in the cells from WT and TRPV2KO mice with different differentiation media. 1/10 suppl. indicates differentiation medium ten-time-diluted with DMEM. Mean ± SEM, n = 8, ** P < 0.01 vs. control group, ##P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Basal mRNA expression of Ucp1 , peroxisome proliferator - activated receptor gamma coactivator 1 - alpha ( Pgc1α ) , peroxisome proliferator - activated receptorγ ( Pparγ ) and β3 - adrenergic receptor 3 ( Adrb3 ) in the differentiated brown adipocytes from WT and TRPV2KO mice . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . WT . Unpaired Student ' s t - test .

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A. Basal mRNA expression of Ucp1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α), peroxisome proliferator-activated receptorγ(Pparγ) and β3-adrenergic receptor 3 (Adrb3) in the differentiated brown adipocytes from WT and TRPV2KO mice. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. WT. Unpaired Student's t-test.


B and C . Changes in Ucp1 ( B ) and Pgc1α ( C ) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol / L isoproterenol ( ISO ) for 4 h . Data are presented as mean ± SEM , n = 5 ; * * P < 0 . 01 vs . DMSO group ; # P < 0 . 05 ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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B and C. Changes in Ucp1 (B) and Pgc1α(C) mRNA expression in the differentiated brown adipocytes from WT and TRPV2KO mice with and without 10 µmol/L isoproterenol (ISO) for 4 h. Data are presented as mean ± SEM, n = 5; ** P < 0.01 vs. DMSO group; # P < 0.05; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


D and E . Changes in Ucp1 mRNA expression ( D ) and non - esterified fatty acid ( NEFA ) release ( E ) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol / L forskolin for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . WT group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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D and E. Changes in Ucp1 mRNA expression (D) and non-esterified fatty acid (NEFA) release (E) in the differentiated brown adipocytes from WT and TRPV2KO mice with or without 10 µmol/L forskolin for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. WT group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


F and G . Changes in Ucp1 mRNA ( F ) and Pgc1α mRNA ( G ) in the differentiated brown adipocytes treated with 10 µmol / L ISO alone or 10 µmol / L ISO plus 10 µmol / L BAPTA - AM for 4 h . Data are presented as mean ± SEM , n = 6 ; * P < 0 . 05 ; * * P < 0 . 01 vs . DMSO group ; # # P < 0 . 01 vs . ISO group . One - way ANOVA followed by 2 - tailed t - test with Bonferroni correction .

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F and G. Changes in Ucp1 mRNA (F) and Pgc1α mRNA (G) in the differentiated brown adipocytes treated with 10 µmol/L ISO alone or 10 µmol/L ISO plus 10 µmol/L BAPTA-AM for 4 h. Data are presented as mean ± SEM, n = 6; * P < 0.05; ** P < 0.01 vs. DMSO group; ## P < 0.01 vs. ISO group. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.


A . Body weight changes from 3 - weeks to 8 - weeks of age between the two genotypes ( n = 11 ) . Data are presented as mean ± SEM ; * P < 0 . 05 ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

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A. Body weight changes from 3-weeks to 8-weeks of age between the two genotypes (n = 11). Data are presented as mean ± SEM; * P < 0.05; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


B and C . Changes in food intake ( B ) and water intake ( C ) from 3 - weeks to 8 - weeks of ages between WT and TRPV2KO mice ( n = 6 ) .

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B and C. Changes in food intake (B) and water intake (C) from 3-weeks to 8-weeks of ages between WT and TRPV2KO mice (n = 6).


D . Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice ( n = 15 ) .

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D. Weights of tissues related to energy metabolism normalized to their body weights in WT and TRPV2KO mice (n = 15).


E . The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice . mRNA expression levels of Ucp1 , Pgc1a , Ppary , Adrb3 , cytochrome c oxidase subunit 4 isoform 1 ( Cox4i1 ) , PR domain containing 16 ( Prdm16 ) , Ucp2 , lipoprotein lipase ( Lpl ) and cluster of differentiation 36 ( Cd36 ) were examined and normalized to the levels of 36B4 . Data are presented as mean ± SEM , n = 15 ; * P < 0 . 05 vs . WT group ; * * P < 0 . 01 vs . WT group . Unpaired Student ' s t - test .

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E. The expression levels of mRNA related to energy metabolism in iBAT from WT and TRPV2KO mice. mRNA expression levels of Ucp1, Pgc1a, Ppary, Adrb3, cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), PR domain containing 16 (Prdm16), Ucp2, lipoprotein lipase (Lpl) and cluster of differentiation 36 (Cd36) were examined and normalized to the levels of 36B4. Data are presented as mean ± SEM, n = 15; * P < 0.05 vs. WT group; ** P < 0.01 vs. WT group. Unpaired Student's t-test.


A . Representative morphological results of iBAT by hematoxylin and eosin staining from 8 - week WT and TRPV2KO mice . Scale bars indicate 100 μm . B . Comparison of lipid droplet size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . Mean values are 9 . 5 ± 0 . 2 µm in 8 - week - old WT adipocytes , and 14 . 7 ± 0 . 2 µm in 8 - week - old TRPV2KO adipocytes ( P < 0 . 01 ) . 1000 to 1400 droplets were counted . Data are presented as mean ± SEM , n = 4 . C . Comparison of adipocyte size histograms from 8 - week - old WT ( black ) and TRPV2KO ( red ) iBAT with hematoxylin and eosin staining . 800 to 1000 cells were counted . Data are presented as mean ± SEM , n = 4 . D . Mean diameters of adipocytes in 8 - week - old WT mice ( black ) and TRPV2KO mice ( red ) . Mean values are 15 . 8 ± 0 . 4 µm in 8 - week - old WT adipocytes , and 19 . 8 ± 0 . 3 µm in 8 - week - old TRPV2KO adipocytes . Data are presented as mean ± SEM , n = 4 ; * P < 0 . 05 vs . WT group . Unpaired Student ' s t - test .

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A. Representative morphological results of iBAT by hematoxylin and eosin staining from 8-week WT and TRPV2KO mice. Scale bars indicate 100 μm.B. Comparison of lipid droplet size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. Mean values are 9.5 ± 0.2 µm in 8-week-old WT adipocytes, and 14.7 ± 0.2 µm in 8-week-old TRPV2KO adipocytes (P < 0.01). 1000 to 1400 droplets were counted. Data are presented as mean ± SEM, n = 4.C. Comparison of adipocyte size histograms from 8-week-old WT (black) and TRPV2KO (red) iBAT with hematoxylin and eosin staining. 800 to 1000 cells were counted. Data are presented as mean ± SEM, n = 4.D. Mean diameters of adipocytes in 8-week-old WT mice (black) and TRPV2KO mice (red). Mean values are 15.8 ± 0.4 µm in 8-week-old WT adipocytes, and 19.8 ± 0.3 µm in 8-week-old TRPV2KO adipocytes. Data are presented as mean ± SEM, n = 4; * P < 0.05 vs. WT group. Unpaired Student's t-test.