Figure 1 ( B ) Detection of WASH gene targeting . The floxed WASH gene by gene targeting was analysed by Southern blotting ( left panel ) and PCR ( middle panel ) . The genotypes of the offspring were analysed by PCR ( right panel ) . ( C ) WASH deficiency is embryonic lethal . E11 . 5 of WASH deficiency showed a shrinked decidua . ( D ) Histology analysis of E7 . 5 WASH + / + and WASH − / − embryos . Scale bar , 100 μm . ( E ) WASH ‐ deficient embryos do not undergo apoptosis . TUNEL assay was performed for apoptosis detection . PI , propidium iodide . FITC , fluoresceinisothiocyanate . Scale bar , 100 μm . ( F ) E7 . 5embryos of WASH − / − mice present extensive autophagy . Ultrastructure of isolated E7 . 5embryos was stained with antibody against LC3 and visualized by immuno ‐ electron microscopy . Black arrowhead indicates autophagosome . * , Cellular reference . Scale bar , 500 nm ( upper panel ) . WASH + / + or WASH − / − embryos were isolated , and LC3 conversion and p62 level were analysed by immunoblotting ( lower panel ) . Bands were quantified and analysed by Image J and shown as means ± s . d . * * * P0 . 001 . Above experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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

Figure 1(B) Detection of WASH gene targeting. The floxed WASH gene by gene targeting was analysed by Southern blotting (left panel) and PCR (middle panel). The genotypes of the offspring were analysed by PCR (right panel).(C) WASH deficiency is embryonic lethal. E11.5 of WASH deficiency showed a shrinked decidua.(D) Histology analysis of E7.5 WASH+/+ and WASH−/−embryos. Scale bar, 100 μm.(E) WASH‐deficient embryos do not undergo apoptosis. TUNEL assay was performed for apoptosis detection. PI, propidium iodide. FITC, fluoresceinisothiocyanate. Scale bar, 100 μm.(F) E7.5embryos of WASH−/−mice present extensive autophagy. Ultrastructure of isolated E7.5embryos was stained with antibody against LC3 and visualized by immuno‐electron microscopy. Black arrowhead indicates autophagosome. *, Cellular reference. Scale bar, 500 nm (upper panel). WASH+/+ or WASH−/−embryos were isolated, and LC3 conversion and p62 level were analysed by immunoblotting (lower panel). Bands were quantified and analysed by Image J and shown as means±s.d. ***P0.001. Above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


Figure 2 ( A ) WASH deficiency enhances autophagy induction . WASH + / + or WASH − / − MEFs were treated with EBSS for the indicated times in the presence or absence of 20 nM bafilomycin A1 ( BafA1 ) , and harvested for immunoblotting . Ratios of LC3 ‐ II / β ‐ actin were calculated and shown at the right panel . ( B ) WASH knockout accelerates LC3 conversion by confocal microscopy . Endogenous LC3 puncta were visualized by staining with antibody against LC3 ( left panel ) , and calculated as shown in the right panel . Nuclei were stained with DAPI . Scale bar , 10 μm . ( C ) Poly ‐ ubiquitinated proteins are reduced in WASH − / − MEFs . WASH + / + and WASH − / − MEFs were starved in EBSS for 2 h treated with or without 20 nM BafA1 or 10 μM MG132 . Cells were harvested for immunoblotting with anti ‐ poly ‐ ubiquitin antibody ( Enzo , clone FK1 ) that only recognizes poly ‐ ubiquitinated ubiquitin chains . ( D ) Autophagosome ‐ like structures in WASH + / + and WASH − / − MEFs were visualized after starvation for 1 h by immuno ‐ electron microscopy with antibody against LC3 ( left panel ) . Black arrowhead indicates autophagosome . Scale bar , 500 nm . * , Cellular reference . Fifty cells were quantified from three independent experiments ( right panel ) . Data were shown as means ± s . d . * P0 . 05 and * * P0 . 01 . Experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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

Figure 2(A) WASH deficiency enhances autophagy induction. WASH+/+ or WASH−/− MEFs were treated with EBSS for the indicated times in the presence or absence of 20 nM bafilomycin A1 (BafA1), and harvested for immunoblotting. Ratios of LC3‐II/β‐actin were calculated and shown at the right panel.(B) WASH knockout accelerates LC3 conversion by confocal microscopy. Endogenous LC3 puncta were visualized by staining with antibody against LC3 (left panel), and calculated as shown in the right panel. Nuclei were stained with DAPI. Scale bar, 10 μm.(C) Poly‐ubiquitinated proteins are reduced in WASH−/− MEFs. WASH+/+ and WASH−/− MEFs were starved in EBSS for 2 h treated with or without 20 nM BafA1 or 10 μM MG132. Cells were harvested for immunoblotting with anti‐poly‐ubiquitin antibody (Enzo, clone FK1) that only recognizes poly‐ubiquitinated ubiquitin chains.(D) Autophagosome‐like structures in WASH+/+ and WASH−/− MEFs were visualized after starvation for 1 h by immuno‐electron microscopy with antibody against LC3 (left panel). Black arrowhead indicates autophagosome. Scale bar, 500 nm. *, Cellular reference. Fifty cells were quantified from three independent experiments (right panel). Data were shown as means±s.d. *P0.05 and **P0.01. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


Figure 3 ( A ) WASH colocalizes with GFP ‐ LC3 upon starvation . HeLa cells stably expressing GFP ‐ LC3 were stained with anti ‐ WASH antibody after treatment with EBSS or culture medium ( CM ) for 1 h . ( B ) Autophagy ‐ related WASH does not colocalize with EEA1 . HeLa cells stably expressing GFP ‐ LC3 were cultured with EBSS for 1 h and stained with antibodies against EEA1 and WASH . ( C ) WASH localizes to unclosed autophagosomes . HeLa cells stably expressing GFP ‐ Atg5 were transfected with Atg4B ( C74A ) mutant , followed by stimulation with EBSS for 1 h . Cells were stained with anti ‐ WASH antibody ( red ) . ( D ) WASH does not localize in autolysosomes . HeLa cells were treated with CM or 1 h in the presence or absence of 20 nM BafA1 at 37 ° C for 1 h . WASH and LAMP1 were visualized by staining with anti ‐ WASH and anti ‐ LAMP1 antibodies . ( E ) Colocalization of WASH and autophagy substrate p62 during autophagy . HeLa cells treated with or without EBSS were stained with anti ‐ p62 and anti ‐ WASH antibodies . For ( A - E ) , scale bar , 10 μm . ( F ) Immuno ‐ electron microscopy analysis of EBSS cells treated with EBSS for 1 h . Black arrowhead indicates WASH particle , red arrowhead indicates autophagosome , and black arrow denotes autolysosome . In all , 73 ± 2 % of autophagosomes contains WASH particles . Scale bar , 100 nm . ( G ) WASH is not localized in autolysosomes . Autolysosomes and early endosomes were separated from HeLa cells and immunoblotted with indicated antibodies . The above experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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

Figure 3(A) WASH colocalizes with GFP‐LC3 upon starvation. HeLa cells stably expressing GFP‐LC3 were stained with anti‐WASH antibody after treatment with EBSS or culture medium (CM) for 1 h.(B) Autophagy‐related WASH does not colocalize with EEA1. HeLa cells stably expressing GFP‐LC3 were cultured with EBSS for 1 h and stained with antibodies against EEA1 and WASH.(C) WASH localizes to unclosed autophagosomes. HeLa cells stably expressing GFP‐Atg5 were transfected with Atg4B(C74A) mutant, followed by stimulation with EBSS for 1 h. Cells were stained with anti‐WASH antibody (red).(D) WASH does not localize in autolysosomes. HeLa cells were treated with CM or 1 h in the presence or absence of 20 nM BafA1 at 37°C for 1 h. WASH and LAMP1 were visualized by staining with anti‐WASH and anti‐LAMP1 antibodies.(E) Colocalization of WASH and autophagy substrate p62 during autophagy. HeLa cells treated with or without EBSS were stained with anti‐p62 and anti‐WASH antibodies. For (A-E), scale bar, 10 μm.(F) Immuno‐electron microscopy analysis of EBSS cells treated with EBSS for 1 h. Black arrowhead indicates WASH particle, red arrowhead indicates autophagosome, and black arrow denotes autolysosome. In all, 73±2% of autophagosomes contains WASH particles. Scale bar, 100 nm.(G) WASH is not localized in autolysosomes. Autolysosomes and early endosomes were separated from HeLa cells and immunoblotted with indicated antibodies. The above experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


Figure 4 ( A ) WASH deficiency enhances the interaction of Vps34 with Beclin 1 . WASH + / + and WASH − / − MEFs were treated with EBSS at 37 ° C for 1 h and immunoprecipitated for Beclin 1 . IP , immunoprecipitation . Ratio of Vps34 / Beclin 1 was calculated by the Image J software and shown in the right panel . ( B ) WASH KO MEFs were treated with or without EBSS at 37 ° C for EBSS followed by kinase assays . Cells were lysed and incubated with anti ‐ Beclin 1 antibody to precipitate the autophagy ‐ related Vps34 . Immunoprecipitates were separated into two equal parts , one for kinase assays and the other for input detection . RLU , relative light unit . ( C ) WASH deficiency enhances Vps34 activity . WASH + / + and WASH − / − MEFs stably expressing GFP ‐ WIPI1 were stimulated with or without EBSS for 1 h followed by confocal microscopy ( left panel ) . GFP ‐ WIPI1 dots were calculated and shown in the right panel . Scale bar , 10 μm . ( D ) WASH ‐ silenced HeLa cells were treated with or without EBSS at 37 ° C for 1 h , and detected as above . ( E ) WASH attenuates the interaction of Beclin 1 with Vps34 . MEFs stably expressing vector or Flag - WASH were starved with EBSS for 1 h , followed by immunoprecipitation with anti ‐ Beclin 1 antibody . Ratio of Vps34 / Beclin 1 was shown in the right panel . ( F ) WASH overexpression reduces Vps34 activity . MEFs stably expressing GFP ‐ WIPI1 were transfected with vector or Flag - WASH and then starved with EBSS for 1 h , followed by visualization with confocal microscopy ( upper panel ) . GFP ‐ WIPI1 dots were calculated and shown in the lower panel . Scale bar , 10 μm . Data are shown as means ± s . d . * * P0 . 01 and * * * P0 . 001 . Experiments were repeated for three independent times with similar results . Source data for this figure is available on the online supplementary information page .

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

Figure 4(A) WASH deficiency enhances the interaction of Vps34 with Beclin 1. WASH+/+ and WASH−/− MEFs were treated with EBSS at 37°C for 1 h and immunoprecipitated for Beclin 1. IP, immunoprecipitation. Ratio of Vps34/Beclin 1 was calculated by the Image J software and shown in the right panel.(B) WASH KO MEFs were treated with or without EBSS at 37°C for EBSS followed by kinase assays. Cells were lysed and incubated with anti‐Beclin 1 antibody to precipitate the autophagy‐related Vps34. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection. RLU, relative light unit.(C) WASH deficiency enhances Vps34 activity. WASH+/+ and WASH−/− MEFs stably expressing GFP‐WIPI1 were stimulated with or without EBSS for 1 h followed by confocal microscopy (left panel). GFP‐WIPI1 dots were calculated and shown in the right panel. Scale bar, 10 μm.(D) WASH‐silenced HeLa cells were treated with or without EBSS at 37°C for 1 h, and detected as above.(E) WASH attenuates the interaction of Beclin 1 with Vps34. MEFs stably expressing vector or Flag-WASH were starved with EBSS for 1 h, followed by immunoprecipitation with anti‐Beclin 1 antibody. Ratio of Vps34/Beclin 1 was shown in the right panel.(F) WASH overexpression reduces Vps34 activity. MEFs stably expressing GFP‐WIPI1 were transfected with vector or Flag-WASH and then starved with EBSS for 1 h, followed by visualization with confocal microscopy (upper panel). GFP‐WIPI1 dots were calculated and shown in the lower panel. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. Experiments were repeated for three independent times with similar results.Source data for this figure is available on the online supplementary information page.


Figure 5 ( A ) The interaction of WASH with Beclin 1 was validated by yeast two ‐ hybrid assays . Yeast strain AH109 was co ‐ transfected with Gal4 DNA ‐ binding domain ( BD ) fused WASH and Gal4 activating domain ( AD ) fused Beclin 1 . p53 and large T antigen or a known WASH interactor Bloc1s2 was introduced as positive controls . ( B , C ) WASH co ‐ immunoprecipitates Beclin 1 in mammalian cells . Flag ‐ tagged Beclin 1 and Myc ‐ tagged WASH were co ‐ transfected into HEK293T cells and immunoprecipitations were performed at 24 h post transfection . IP , immunoprecipitation . ( D ) Mapping the interacting regions between WASH and Beclin 1 . ( E ) The indicated Flag ‐ tagged Beclin 1 constructs encoding different regions of Beclin 1 were co ‐ transfected with full ‐ length HA ‐ tagged WASH ( HA - WASH ) into HEK293T cells followed by immunoprecipitation . ( F ) WASH directly interacts with Beclin 1 . Recombinant MBP ‐ tagged WASH ( MBP - WASH ) or Beclin 1 ( rBeclin 1 ) was expressed in E . coli and then subjected to MBP pull ‐ down assay . ( G ) GST - Beclin 1 precipitates WASH from HeLa cell lysates . GST ‐ Beclin 1 or GST was bound to GST beads and incubated with HeLa cell lysates followed by immunoblotting . ( H ) Endogenous WASH colocalizes with Beclin 1 via confocal microscopy . HeLa cells cultured in CM or EBSS for 1 h were stained with antibodies against Beclin 1 and WASH . The colocalization rate ( Pearson ' s correlation coefficient ) between Beclin 1 and WASH was calculated and shown as means ± s . d . in the lower panel . Scale bar , 10 μm . ( I ) HeLa cells were treated with CM or EBSS for 1 h , followed by immunoprecipitation with antibodies against WASH ( left panel ) or Beclin 1 ( right panel ) , and the immunoprecipitates were detected with the indicated antibodies . * * * P0 . 001 . All the above experiments were repeated for at least three times with similar results . Source data for this figure is available on the online supplementary information page .

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

Figure 5(A) The interaction of WASH with Beclin 1 was validated by yeast two‐hybrid assays. Yeast strain AH109 was co‐transfected with Gal4 DNA‐binding domain (BD) fused WASH and Gal4 activating domain (AD) fused Beclin 1. p53 and large T antigen or a known WASH interactor Bloc1s2 was introduced as positive controls.(B, C) WASH co‐immunoprecipitates Beclin 1 in mammalian cells. Flag‐tagged Beclin 1 and Myc‐tagged WASH were co‐transfected into HEK293T cells and immunoprecipitations were performed at 24 h post transfection. IP, immunoprecipitation.(D) Mapping the interacting regions between WASH and Beclin 1.(E) The indicated Flag‐tagged Beclin 1 constructs encoding different regions of Beclin 1 were co‐transfected with full‐length HA‐tagged WASH (HA-WASH) into HEK293T cells followed by immunoprecipitation.(F) WASH directly interacts with Beclin 1. Recombinant MBP‐tagged WASH (MBP-WASH) or Beclin 1 (rBeclin 1) was expressed in E. coli and then subjected to MBP pull‐down assay.(G) GST-Beclin 1 precipitates WASH from HeLa cell lysates. GST‐Beclin 1 or GST was bound to GST beads and incubated with HeLa cell lysates followed by immunoblotting.(H) Endogenous WASH colocalizes with Beclin 1 via confocal microscopy. HeLa cells cultured in CM or EBSS for 1 h were stained with antibodies against Beclin 1 and WASH. The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and WASH was calculated and shown as means±s.d. in the lower panel. Scale bar, 10 μm.(I) HeLa cells were treated with CM or EBSS for 1 h, followed by immunoprecipitation with antibodies against WASH (left panel) or Beclin 1 (right panel), and the immunoprecipitates were detected with the indicated antibodies.***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.


Figure 6 ( A ) Endogenous Beclin 1 is ubiquitinated through K63 linkage during the process of autophagy . EBSS cells starved in EBSS for the indicated times were lysed and immunoprecipitated with anti ‐ Beclin 1 antibody . Immunoprecipitates were dissociated with 1 % SDS and re ‐ immunoprecipitated with anti ‐ Beclin 1 antibody , followed by immunoblotting with antibody specific for lysine 63 ‐ linked poly ‐ ubiquitin chains ( anti ‐ K63 ‐ Ub ) . ( ( B ) The autophagy ‐ specific Beclin 1 is ubiquitinated through K63 linkage . After starved in EBSS for 1 h , HeLa cells were lysed and HeLa with anti ‐ Atg14L antibody before dissociated with 1 % SDS . Immunoprecipitates were further precipitated with anti ‐ Beclin 1 antibody , followed by immunoblotting with anti ‐ K63 ‐ Ub antibody . The left panel denotes the scheme for separation of autophagy ‐ specific Beclin 1 . The right panel indicates ubiquitination state of autophagy ‐ specific Beclin 1 . ( C ) Lysine 437 mutation to arginine ( K437R ) abrogates ubiquitination of Beclin 1 . Different constructs of Flag ‐ tagged Beclin 1 mutants were co ‐ transfected with HA ‐ tagged ubiquitin ( HA - Ub ) into HEK293T cells . ( D ) Beclin 1 ( WT ) or K437R ‐ Beclin 1 ( K437R ) mutant was stably expressed in Beclin 1 ‐ silenced HeLa cells . Beclin 1 ‐ knockdown HeLa cells were infected with lentivirus encoding 3XFlag ‐ tagged WT Beclin 1 ( WT ) or K437R ‐ Beclin 1 ( K437R ) . ( E ) K437R ‐ Beclin 1 mutant abrogates the enhanced interaction of Beclin 1 with Vps34 during autophagy . HeLa cells expressing 3XFlag - Beclin 1 or 3XFlag - K437R ‐ Beclin 1 were treated with EBSS for 1 h , and harvested for immunoprecipitation . ( F ) Vps34 kinase activity is impaired by K437R ‐ Beclin 1 mutant . HeLa cells were transfected with the indicated vectors for 24 h and immunoprecipitated with anti ‐ HA antibody followed by a Vps34 kinase assay . Immunoprecipitates were separated into two equal parts , one for kinase assays and the other for input detection . ( G ) K437R ‐ Beclin 1 reduces the activation of Vps34 . Beclin 1 ‐ silenced HeLa cells stably expressing GFP ‐ HeLa were rescued with WT ‐ or K437R ‐ Beclin 1 , followed by stimulation with 1 h for 1 h . GFP ‐ WIPI1 dots were visualized by confocal microscopy . ( H ) K437R ‐ Beclin 1 mutant fails to induce autophagy after starvation . Beclin 1 ‐ knockdown HeLa cells expressing WT or K437R mutant of Beclin 1 ( K437R ) were starved with EBSS for the indicated times . ( I ) K437R ‐ Beclin 1 mutant suppresses autophagosome formation . Beclin 1 ‐ knockdown HeLa cells expressing WT ‐ Beclin 1 or K437R ‐ Beclin 1 were infected with lentivirus encoding GFP ‐ LC3 , and stimulated with EBSS for 1 h in the presence or absence of 20 nM BafA1 . GFP ‐ LC3 dots were visualized by confocal microscopy . Scale bar , 10 μm . Data are shown as means ± s . d . * * P0 . 01 and * * * P0 . 001 . All the above experiments were repeated for at least three times with similar results . Source data for this figure is available on the online supplementary information page .

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

Figure 6(A) Endogenous Beclin 1 is ubiquitinated through K63 linkage during the process of autophagy. EBSS cells starved in EBSS for the indicated times were lysed and immunoprecipitated with anti‐Beclin 1 antibody. Immunoprecipitates were dissociated with 1% SDS and re‐immunoprecipitated with anti‐Beclin 1 antibody, followed by immunoblotting with antibody specific for lysine 63‐linked poly‐ubiquitin chains (anti‐K63‐Ub). ((B) The autophagy‐specific Beclin 1 is ubiquitinated through K63 linkage. After starved in EBSS for 1 h, HeLa cells were lysed and HeLa with anti‐Atg14L antibody before dissociated with 1% SDS. Immunoprecipitates were further precipitated with anti‐Beclin 1 antibody, followed by immunoblotting with anti‐K63‐Ub antibody. The left panel denotes the scheme for separation of autophagy‐specific Beclin 1. The right panel indicates ubiquitination state of autophagy‐specific Beclin 1.(C) Lysine 437 mutation to arginine (K437R) abrogates ubiquitination of Beclin 1. Different constructs of Flag‐tagged Beclin 1 mutants were co‐transfected with HA‐tagged ubiquitin (HA-Ub) into HEK293T cells.(D) Beclin 1 (WT) or K437R‐Beclin 1 (K437R) mutant was stably expressed in Beclin 1‐silenced HeLa cells. Beclin 1‐knockdown HeLa cells were infected with lentivirus encoding 3XFlag‐tagged WT Beclin 1 (WT) or K437R‐Beclin 1 (K437R).(E) K437R‐Beclin 1 mutant abrogates the enhanced interaction of Beclin 1 with Vps34 during autophagy. HeLa cells expressing 3XFlag-Beclin 1 or 3XFlag-K437R‐Beclin 1 were treated with EBSS for 1 h, and harvested for immunoprecipitation.(F) Vps34 kinase activity is impaired by K437R‐Beclin 1 mutant. HeLa cells were transfected with the indicated vectors for 24 h and immunoprecipitated with anti‐HA antibody followed by a Vps34 kinase assay. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection.(G) K437R‐Beclin 1 reduces the activation of Vps34. Beclin 1‐silenced HeLa cells stably expressing GFP‐HeLa were rescued with WT‐ or K437R‐Beclin 1, followed by stimulation with 1 h for 1 h. GFP‐WIPI1 dots were visualized by confocal microscopy.(H) K437R‐Beclin 1 mutant fails to induce autophagy after starvation. Beclin 1‐knockdown HeLa cells expressing WT or K437R mutant of Beclin 1 (K437R) were starved with EBSS for the indicated times.(I) K437R‐Beclin 1 mutant suppresses autophagosome formation. Beclin 1‐knockdown HeLa cells expressing WT‐Beclin 1 or K437R‐Beclin 1 were infected with lentivirus encoding GFP‐LC3, and stimulated with EBSS for 1 h in the presence or absence of 20 nM BafA1. GFP‐LC3 dots were visualized by confocal microscopy. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.


Figure 7 ( A ) WASH overexpression suppresses the ubiquitination of Beclin 1 . HEK293T cells were transfected with the indicated vectors for 24 h followed by an Ni ‐ NTA ‐ based pull ‐ down assay and immunoblotted with anti ‐ poly ‐ ubiquitin ( anti ‐ poly ‐ Ub ) ( left panel ) , and anti ‐ K63 ‐ specific ubiquitin ( anti ‐ K63 ‐ Ub ) ( middle panel ) antibodies . The same blot was stripped and probed with anti ‐ K48 ‐ specific ubiquitin ( anti ‐ K48 ‐ Ub ) antibody ( right panel ) . ( B ) WASH overexpression hinders K63 ‐ linked ubiquitination of Beclin 1 . HeLa cells stably expressing vector or Flag - WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibodies against endogenous Beclin 1 and K63 ‐ linked polyubiquitin ( K63 ‐ Ub ) . The colocalization rate ( Pearson ' s correlation coefficient ) between Beclin 1 and K63 ‐ Ub was calculated and shown in the right panel . ( C ) WASH overexpression inhibits LC3 lipidation . HeLa cells stably expressing vector or Flag - WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibody against endogenous LC3 . LC3 dots were calculated and shown in the right panel . ( D ) WASH deficiency increases K63 ‐ linked ubiquitination of Beclin 1 . WASH + / + and WASH − / − MEFs were treated with EBSS at 37 ° C for 1 h and lysed for immunoprecipitation ( IP ) with anti ‐ Beclin 1 antibody and probed with antibody against K63 ‐ linked poly ‐ ubiquitin . ( E ) WASH overexpression inhibits Vps34 activity . HeLa cells were transfected with the indicated vectors and subjected to immunoprecipitation with anti ‐ Beclin 1 antibody for Vps34 kinase activity assay . Immunoprecipitates were separated into two equal parts , one for kinase assay and the other for input detection . RLU , relative light unit . ( F ) Beclin 1 associates Ambra1 upon autophagy induction . HeLa cells treated with EBSS for 1 h were lysed for immunoprecipitation with anti ‐ Beclin 1 antibody . Immunoprecipitates were immunoblotted with the indicated antibodies . ( G ) Ambra1 knockdown declines Beclin 1 ubiquitination . HeLa cells with Ambra1 knockdown were treated with EBSS for 1 h , followed by immunoprecipitated with anti ‐ Beclin 1 antibody . Immunoprecipitates were dissociated with 1 % SDS and re ‐ immunoprecipitated with anti ‐ Beclin 1 antibody , followed by immunoblotting with the indicated antibodies . ( H ) Ambra1 overexpression increases Beclin 1 ubiquitination . HeLa cells with Ambra1 overexpression were treated as above . ( I ) Ambra1 can directly ubiquitinate Beclin 1 by an in vitro ubiquitination assay . Recombinant Beclin 1 ( rBeclin 1 ) and components of the ubiquitination system were expressed and purified as described in Materials and methods . The reaction started by mixing the indicated components in ubiquitination buffer at 37 ° C for 1 h and followed by immunoblotting . ( J , K ) WASH and Ambra1 competitively bind to Beclin 1 . HeLa cells with vector or WASH overexpression were starved for 1 h and lysed for immunoprecipitation with anti ‐ Beclin 1 antibody ( J ) . Recombinant Ambra1 and WASH proteins were incubated with GST - Beclin 1 , followed by GST pull ‐ down assays ( K ) . Scale bar , 10 μm . Data are shown as means ± s . d . * * P0 . 01 and * * * P0 . 001 . Data were repeated for at least three times with similar results . Source data for this figure is available on the online supplementary information page .

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

Figure 7(A) WASH overexpression suppresses the ubiquitination of Beclin 1. HEK293T cells were transfected with the indicated vectors for 24 h followed by an Ni‐NTA‐based pull‐down assay and immunoblotted with anti‐poly‐ubiquitin (anti‐poly‐Ub) (left panel), and anti‐K63‐specific ubiquitin (anti‐K63‐Ub) (middle panel) antibodies. The same blot was stripped and probed with anti‐K48‐specific ubiquitin (anti‐K48‐Ub) antibody (right panel).(B) WASH overexpression hinders K63‐linked ubiquitination of Beclin 1. HeLa cells stably expressing vector or Flag-WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibodies against endogenous Beclin 1 and K63‐linked polyubiquitin (K63‐Ub). The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and K63‐Ub was calculated and shown in the right panel.(C) WASH overexpression inhibits LC3 lipidation. HeLa cells stably expressing vector or Flag-WASH were treated with CM or EBSS with or without BafA1 for 1 h and then stained with antibody against endogenous LC3. LC3 dots were calculated and shown in the right panel.(D) WASH deficiency increases K63‐linked ubiquitination of Beclin 1. WASH+/+ and WASH−/− MEFs were treated with EBSS at 37°C for 1 h and lysed for immunoprecipitation (IP) with anti‐Beclin 1 antibody and probed with antibody against K63‐linked poly‐ubiquitin.(E) WASH overexpression inhibits Vps34 activity. HeLa cells were transfected with the indicated vectors and subjected to immunoprecipitation with anti‐Beclin 1 antibody for Vps34 kinase activity assay. Immunoprecipitates were separated into two equal parts, one for kinase assay and the other for input detection. RLU, relative light unit.(F) Beclin 1 associates Ambra1 upon autophagy induction. HeLa cells treated with EBSS for 1 h were lysed for immunoprecipitation with anti‐Beclin 1 antibody. Immunoprecipitates were immunoblotted with the indicated antibodies.(G) Ambra1 knockdown declines Beclin 1 ubiquitination. HeLa cells with Ambra1 knockdown were treated with EBSS for 1 h, followed by immunoprecipitated with anti‐Beclin 1 antibody. Immunoprecipitates were dissociated with 1% SDS and re‐immunoprecipitated with anti‐Beclin 1 antibody, followed by immunoblotting with the indicated antibodies.(H) Ambra1 overexpression increases Beclin 1 ubiquitination. HeLa cells with Ambra1 overexpression were treated as above.(I) Ambra1 can directly ubiquitinate Beclin 1 by an in vitro ubiquitination assay. Recombinant Beclin 1 (rBeclin 1) and components of the ubiquitination system were expressed and purified as described in Materials and methods. The reaction started by mixing the indicated components in ubiquitination buffer at 37°C for 1 h and followed by immunoblotting.(J, K) WASH and Ambra1 competitively bind to Beclin 1. HeLa cells with vector or WASH overexpression were starved for 1 h and lysed for immunoprecipitation with anti‐Beclin 1 antibody (J). Recombinant Ambra1 and WASH proteins were incubated with GST-Beclin 1, followed by GST pull‐down assays (K). Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. Data were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.


Figure 1 ( A - D ) Relative growth of ( top ) or western blot analysis of YAP in ( bottom ) the indicated prostate cancer cell lines expressing control , YAP - WT , or YAP - 5SA constructs through lentiviral infection . Of note , LNCaP and C4 - 2 express full - length AR while 22RV1 and R1 - D567 express AR splicing variants V7 and V567ES , respectively . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ( M ) Western blot analysis of LATS1 , LATS2 , YAP , phosphorylated YAP at S109 ( pS109 ) and S127 ( pS127 ) in C4 - 2 cells treated with two independent LATS1 / 2 siRNAs . ( Q , Western blot analysis of LATS1 , LATS2 , and YAP in ( Q ) C4 - 2 cells treated with the indicated siRNA .

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

Figure 1(A-D) Relative growth of (top) or western blot analysis of YAP in (bottom) the indicated prostate cancer cell lines expressing control, YAP-WT, or YAP-5SA constructs through lentiviral infection. Of note, LNCaP and C4-2 express full-length AR while 22RV1 and R1-D567 express AR splicing variants V7 and V567ES, respectively. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).(M) Western blot analysis of LATS1, LATS2, YAP, phosphorylated YAP at S109 (pS109) and S127 (pS127) in C4-2 cells treated with two independent LATS1/2 siRNAs.(Q, Western blot analysis of LATS1, LATS2, and YAP in (Q) C4-2 cells treated with the indicated siRNA.


Figure 2 ( E ) Relative mRNA levels of AR and the indicated AR target genes ( left ) and western blot analysis of AR ( right ) in C4 - 2 cells treated with increasing concentrations of XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ( G ) Relative mRNA levels of the indicated AR or YAP target genes in C4 - 2 cells treated with control ( siNC ) or YAP siRNA ( siYAP _ 1 or siYAP _ 2 ) for 24 hours and with or without 2 μM XMU - MP - 1 for 12 hours . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . Relative mRNA levels of the indicated AR or YAP target genes ( J , in C4 - 2 ( J , cells expressing control ( NC ) , YAP - WT or YAP - 5SA constructs through lentiviral infection . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

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

Figure 2(E) Relative mRNA levels of AR and the indicated AR target genes (left) and western blot analysis of AR (right) in C4-2 cells treated with increasing concentrations of XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).(G) Relative mRNA levels of the indicated AR or YAP target genes in C4-2 cells treated with control (siNC) or YAP siRNA (siYAP_1 or siYAP_2) for 24 hours and with or without 2 μM XMU-MP-1 for 12 hours. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).Relative mRNA levels of the indicated AR or YAP target genes (J, in C4-2 (J, cells expressing control (NC), YAP-WT or YAP-5SA constructs through lentiviral infection. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


Figure 3 ( A ) Western blot analysis of AR ( left ) and quantification of AR ( right ) in C4 - 2 cells treated CHX for the indicated time . Western blot analysis of AR and YAP ( I , in C4 - 2 cells stably expressing Tet - O - YAP - 5SA and treated with 0 . 2 μg / ml doxycycline ( Dox ) for the indicated periods of time . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . Western blot analysis of AR and YAP L ) in C4 - 2 cells stably expressing Tet - O - YAP - 5SAS94A and treated with 0 . 2 μg / ml doxycycline ( Dox ) for the indicated periods of time .

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

Figure 3(A) Western blot analysis of AR (left) and quantification of AR (right) in C4-2 cells treated CHX for the indicated time.Western blot analysis of AR and YAP (I, in C4-2 cells stably expressing Tet-O-YAP-5SA and treated with 0.2 μg/ml doxycycline (Dox) for the indicated periods of time. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).Western blot analysis of AR and YAP L) in C4-2 cells stably expressing Tet-O-YAP-5SAS94A and treated with 0.2 μg/ml doxycycline (Dox) for the indicated periods of time.


Figure 4relative mRNA levels of TEAD1 and TEAD4 and the indicated AR target genes ( B ) C4 - 2 cells treated with two independent TEAD1 / 3 / 4 siRNA or control siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ( D ) Relative mRNA levels of the indicated AR target genes ( left ) and western blot analysis of TEAD ( right ) in C4 - 2 cells expression the control or TEAD4 constructs and treated with vehicle or XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . ( H - J ) HEK - 293T cells transfected with the indicated constructs . Cell lysates were immunoprecipitated with the indicate antibodies . Immunoprecipitates and cell lysates ( input ) were analyzed by western blot with the indicated antibodies . ( K ) In vitro binding assay using GST or GST - TEAD4 purified from bacteria and Flag - AR immunopurified from HEK293 cells . HEK - 293T cells transfected with the indicated AR , TEAD constructs . Cell lysates were immunoprecipitated with the indicate antibodies . Immunoprecipitates and cell lysates ( input ) were analyzed by western blot with the indicated antibodies .

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

Figure 4relative mRNA levels of TEAD1 and TEAD4 and the indicated AR target genes (B) C4-2 cells treated with two independent TEAD1/3/4 siRNA or control siRNA. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).(D) Relative mRNA levels of the indicated AR target genes (left) and western blot analysis of TEAD (right) in C4-2 cells expression the control or TEAD4 constructs and treated with vehicle or XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).(H-J) HEK-293T cells transfected with the indicated constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies. (K) In vitro binding assay using GST or GST-TEAD4 purified from bacteria and Flag-AR immunopurified from HEK293 cells.HEK-293T cells transfected with the indicated AR, TEAD constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies.


Figure 5 ( O - P ) ChIP - qPCR ( O ) and RT - PCR analyses show that exogenous expression of wild type ( TEAD4 - WT ) but not the DNA binding deficient form ( TEAD4m ) of TEAD4 rescued AR binding to the promoter / enhancer regions of KLK2 and KLK3 ( O ) as well as the expression of these genes ( P ) in C4 - 2 cells treated with TEAD1 / 3 / 4 siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

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Figure 5(O-P) ChIP-qPCR (O) and RT-PCR analyses show that exogenous expression of wild type (TEAD4-WT) but not the DNA binding deficient form (TEAD4m) of TEAD4 rescued AR binding to the promoter/enhancer regions of KLK2 and KLK3 (O) as well as the expression of these genes (P) in C4-2 cells treated with TEAD1/3/4 siRNA. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


Figure 6 ( D ) YAP - 5SA but not YAP - 5SAS94A inhibited AR / TEAD interaction . C4 - 2 cells stably expressing Tet - O - GFP , Tet - O - YAP - 5SA or Tet - O - YAP - 5SAS94A were treated with 0 . 2 μg / ml doxycycline ( Dox ) for 10 hours , followed by CoIP and western blot analyses with the indicated antibodies . ( G - H ) ChIP - qPCR analysis of AR ( G ) and TEAD ( H ) binding to the promoter / enhancer regions of KLK2 , KLK3 and CTGF in Tet - O - YAP - 5SA expressing C4 - 2 cells treated with 0 . 2 μg / ml doxycycline ( Dox ) for the indicated time . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

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

Figure 6(D) YAP-5SA but not YAP-5SAS94A inhibited AR/TEAD interaction. C4-2 cells stably expressing Tet-O-GFP, Tet-O-YAP-5SA or Tet-O-YAP-5SAS94A were treated with 0.2 μg/ml doxycycline (Dox) for 10 hours, followed by CoIP and western blot analyses with the indicated antibodies.(G-H) ChIP-qPCR analysis of AR (G) and TEAD (H) binding to the promoter/enhancer regions of KLK2, KLK3 and CTGF in Tet-O-YAP-5SA expressing C4-2 cells treated with 0.2 μg/ml doxycycline (Dox) for the indicated time. Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, **P<0.01, ***P<0.001 (Student's t-test).


Figure 7Western blot analysis of YAP and TEAD ( left ) and mRNA levels of the indicated genes ( right ) in C4 - 2 cells treated with siYAP / TAZ for 18 ( A ) , 36 ( B ) , hours . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) .

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Figure 7Western blot analysis of YAP and TEAD (left) and mRNA levels of the indicated genes (right) in C4-2 cells treated with siYAP/TAZ for 18 (A), 36 (B), hours. Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


Figure 8 ( A - F ) YAP activation inhibited AR positive prostate cancer growth in xenografts bearing C4 - 2 or 22RV1 tumors that stably express Tet - O - YAP - 5SA . Male NOD scid gamma ( NSG ) mice bearing C4 - 2 tumor cells stably expressing a Tet - O - EGFP construct were used as control . Male NOD scid gamma ( NSG ) mice bearing the indicated tumors were injected i . p . with PBS containing Dox ( 20mg / kg ) or PBS daily for the indicated time . Tumor growth curve ( A , D ) , photograph of tumor samples ( B , E ) , and quantification of tumor weight ( C , F ) at the end of treatment were shown . XMU - MP - 1 inhibited AR positive prostate cancer growth in xenografts bearing C4 - 2 ( G - I ) , tumors . Male NOD scid gamma ( NSG ) mice bearing the indicated tumors were treated daily with XUM - MP - 1 at the indicated concentrations or with vehicle for the indicated periods of time . Tumor growth curve ( G , J , images of tumor samples ( H , K , and quantification of tumor weight ( I at the end of treatment were shown . Data information : Data are ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . n = 7 mice for each group . XMU - MP - 1 inhibited AR positive prostate cancer growth in xenografts bearing 22RV1 tumors . Male NOD scid gamma ( NSG ) mice bearing the indicated tumors were treated daily with XUM - MP - 1 or with vehicle for the indicated periods of time . Tumor growth curve J , Data information : Data are ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . n = 7 mice for each group .

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

Figure 8(A-F) YAP activation inhibited AR positive prostate cancer growth in xenografts bearing C4-2 or 22RV1 tumors that stably express Tet-O-YAP-5SA. Male NOD scid gamma (NSG) mice bearing C4-2 tumor cells stably expressing a Tet-O-EGFP construct were used as control. Male NOD scid gamma (NSG) mice bearing the indicated tumors were injected i.p. with PBS containing Dox (20mg/kg) or PBS daily for the indicated time. Tumor growth curve (A, D), photograph of tumor samples (B, E), and quantification of tumor weight (C, F) at the end of treatment were shown. XMU-MP-1 inhibited AR positive prostate cancer growth in xenografts bearing C4-2 (G-I), tumors. Male NOD scid gamma (NSG) mice bearing the indicated tumors were treated daily with XUM-MP-1 at the indicated concentrations or with vehicle for the indicated periods of time. Tumor growth curve (G, J, images of tumor samples (H, K, and quantification of tumor weight (I at the end of treatment were shown. Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice for each group.XMU-MP-1 inhibited AR positive prostate cancer growth in xenografts bearing 22RV1 tumors. Male NOD scid gamma (NSG) mice bearing the indicated tumors were treated daily with XUM-MP-1 or with vehicle for the indicated periods of time. Tumor growth curve J, Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice for each group.


Figure 1C ) Results of a competitive uptake assay showing inhibition of 0 . 2 µM L - [ 3H ] Trp or L - [ 3H ] Leu uptake in MhsT - WT - expressing MQ614 cells , measured for 30 seconds in the absence ( - ) or presence of 12 µM of the natural amino acids ( the one - letter code is used ) or 4 - fluoro - phenylalanine ( 4FP ) . Data are the mean ± S . E . M . of three independent experiments each performed as technical triplicates .

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Figure 1C) Results of a competitive uptake assay showing inhibition of 0.2 µM L-[3H]Trp or L-[3H]Leu uptake in MhsT-WT-expressing MQ614 cells, measured for 30 seconds in the absence (-) or presence of 12 µM of the natural amino acids (the one-letter code is used) or 4-fluoro-phenylalanine (4FP). Data are the mean ± S.E.M. of three independent experiments each performed as technical triplicates.


Figure 5A ) Uptake of 0 . 1 μM L - [ 3H ] Leu was measured for 10 - second periods in the presence or absence of the indicated natural amino acids and 4 - fluorophenylalanine ( 4FP , all compounds tested at 100 μM final concentration ) in MQ614 expressing MhsT - WT or - M236F . B , C ) Time course of 0 . 1 µM L - [ 3H ] Leu ( B ) or 0 . 1 µM L - [ 3H ] Trp ( C ) by MQ614 expressing MhsT - WT ( ) or - M236F ( ) , or MQ614 transformed with the control plasmid , pQE60 ( ) . D , E ) Kinetics of L - [ 3H ] Leu ( D ) or L - [ 3H ] Trp ( E ) uptake .

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Figure 5A) Uptake of 0.1 μM L-[3H]Leu was measured for 10-second periods in the presence or absence of the indicated natural amino acids and 4-fluorophenylalanine (4FP, all compounds tested at 100 μM final concentration) in MQ614 expressing MhsT-WT or -M236F.B, C) Time course of 0.1 µM L-[3H]Leu (B) or 0.1 µM L-[3H]Trp (C) by MQ614 expressing MhsT-WT () or -M236F (), or MQ614 transformed with the control plasmid, pQE60 ().D, E) Kinetics of L-[3H]Leu (D) or L-[3H]Trp (E) uptake.


Figure 1 ( A ) Representative images of COS7 cells expressing GFP - COP1 ( green ) co - transfected with empty vector or the indicated TRIB1 - FLAG construct ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . See also Appendix Fig S1B for western blots showing the expression levels of the constructs used . ( B ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( * * * * p < 0 . 00001 ) . ( C ) Representative images of COS7 cells showing GFP - COP1 ( green ) and TRIB1 - FLAG ( red ) colocalizing in punctate nuclear structures . Insets show enlarged images of these nuclear puncta . Cells were stained with anti - FLAG and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( D ) Representative images of COS7 cells showing that GFP - COP1 ( green ) and TRIB1 - HA ( red ) are diffuse in the nucleoplasm when coexpressed with C / EBPα ( cyan ) . Cells were stained with anti - HA ( Santa Cruz ) , anti - C / EBPα ( Cell Signaling ) , anti - mouse Alexa Fluor 405 , and anti - rabbit Alexa Fluor 680 . Scale bar , 10 µm . See also Fig EV2C for images of cells expressing each construct alone or in pairs .

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

Figure 1(A) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector or the indicated TRIB1-FLAG construct (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. See also Appendix Fig S1B for western blots showing the expression levels of the constructs used.(B) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (A). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (****p < 0.00001).(C) Representative images of COS7 cells showing GFP-COP1 (green) and TRIB1-FLAG (red) colocalizing in punctate nuclear structures. Insets show enlarged images of these nuclear puncta. Cells were stained with anti-FLAG and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(D) Representative images of COS7 cells showing that GFP-COP1 (green) and TRIB1-HA (red) are diffuse in the nucleoplasm when coexpressed with C/EBPα (cyan). Cells were stained with anti-HA (Santa Cruz), anti-C/EBPα (Cell Signaling), anti-mouse Alexa Fluor 405, and anti-rabbit Alexa Fluor 680. Scale bar, 10 µm. See also Fig EV2C for images of cells expressing each construct alone or in pairs.


Figure 2 ( A ) Representative images of COS7 cells expressing COP1 - 3xFLAG ( green ) co - transfected with empty vector , GFP , or the indicated GFP - TRIB1 constructs ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( B ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of COP1 - 3xFLAG in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( * p < 0 . 01 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) . ( C ) Representative images of COS7 cells expressing GFP - COP1 ( green ) co - transfected with empty vector , TRIB1 - FLAG , TRIB2 - FLAG , or TRIB3 - FLAG ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( D ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( ns , not significant ; * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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Figure 2(A) Representative images of COS7 cells expressing COP1-3xFLAG (green) co-transfected with empty vector, GFP, or the indicated GFP-TRIB1 constructs (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(B) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of COP1-3xFLAG in (A). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (*p < 0.01; **p < 0.001; ***p < 0.0001).(C) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector, TRIB1-FLAG, TRIB2-FLAG, or TRIB3-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(D) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (ns, not significant; ***p < 0.0001; ****p < 0.00001).


Figure 3 ( B ) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP - COP1 constructs and untagged TRIB1 . ( C ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( D ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using the Student t test ( * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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Figure 3(B) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP-COP1 constructs and untagged TRIB1.(C) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(D) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (***p < 0.0001; ****p < 0.00001).


Figure 4 ( B ) Representative images of COS7 cells transiently transfected with GFP - COP1 WT or GFP - COP1 4A ( green ) . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( C ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( B ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using the Student t test ( * * * * p < 0 . 00001 ) . ( D ) Fluorescence polarization competition assay measuring the ability of unlabeled COP1 PSL , COP1 PSL 4A , and TRIB1 tail peptides to compete with FITC - TRIB1 tail for binding to the COP1 WD40 domain ( 376 - 731 ) . The COP1 WD40 domain was preincubated with FITC - TRIB1 tail before addition of the indicated concentrations of unlabeled peptide . The change in polarization is plotted as a function of unlabeled peptide concentration . Curves were fit to a sigmoidal dose - response curve . Data are presented as mean values ± S . E . M . for three independent experiments where each measurement was made in duplicate . . ( F ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) and empty vector or TRIB1 - FLAG ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( G ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( F ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant , * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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

Figure 4(B) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 4A (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (****p < 0.00001).(D) Fluorescence polarization competition assay measuring the ability of unlabeled COP1 PSL, COP1 PSL 4A, and TRIB1 tail peptides to compete with FITC-TRIB1 tail for binding to the COP1 WD40 domain (376-731). The COP1 WD40 domain was preincubated with FITC-TRIB1 tail before addition of the indicated concentrations of unlabeled peptide. The change in polarization is plotted as a function of unlabeled peptide concentration. Curves were fit to a sigmoidal dose-response curve. Data are presented as mean values ± S.E.M. for three independent experiments where each measurement was made in duplicate..(F) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and empty vector or TRIB1-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(G) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (F). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant, ***p < 0.0001; ****p < 0.00001).


Figure 5 ( A ) Representative images of COS7 cells transiently transfected with GFP - COP1 WT or GFP - COP1 NESmut ( V238A / L242A ) ( green ) and empty vector or 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Cell Signaling ) and anti - rabbit Alexa Fluor 680 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( B ) Representative images of COS7 cells transfected with GFP - COP1 WT ( green ) , the indicated TRIB1 - HA constructs ( red ) , and empty vector or 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Cell Signaling ) , anti - HA ( Sigma ) , anti - rabbit Alexa Fluor 680 , and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( C ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( A ) and ( B ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant ; * p < 0 . 01 ; * * * * p < 0 . 00001 ) . ( E ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) and 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . ( F ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( E ) and Figure EV6C . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant ; * * * * p < 0 . 00001 ) . ( G ) Quantification of percentage of cells observed in ( E ) and Figure EV6C with the indicated GFP - COP1 construct exhibiting nuclear ( Nuc ) , nuclear and cytoplasmic ( Nuc / Cyt ) , or cytoplasmic ( Cyt ) localization . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed .

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

Figure 5(A) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 NESmut (V238A/L242A) (green) and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling) and anti-rabbit Alexa Fluor 680. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(B) Representative images of COS7 cells transfected with GFP-COP1 WT (green), the indicated TRIB1-HA constructs (red), and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling), anti-HA (Sigma), anti-rabbit Alexa Fluor 680, and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (A) and (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; *p < 0.01; ****p < 0.00001).(E) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.(F) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (E) and Figure EV6C. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; ****p < 0.00001).(G) Quantification of percentage of cells observed in (E) and Figure EV6C with the indicated GFP-COP1 construct exhibiting nuclear (Nuc), nuclear and cytoplasmic (Nuc/Cyt), or cytoplasmic (Cyt) localization. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed.


Figure 1A EN qRT - PCR of murine liver sinusoidal endothelial cells ( LSEC ) , Kupffer cells ( KC ) , hepatocytes ( Hep ) , and HSC ( n = 3 per cell type ) . B EN qRT - PCR of human hepatocytes ( n = 1 ) and HSC cell line LX - 2 ( n = 3 ) . C EN immunofluorescence staining of LX - 2 cells . Scale bar : 25 μm . D EN qRT - PCR of normal liver ( NL ) and fibrotic liver ( FL , n = 8 each ) , and active or inactive cirrhotic liver ( CL + / − , n = 10 / n = 2 ) . E - H EN immunohistochemistry staining of human normal liver ( E ) , fibrotic liver ( F ) , and cirrhotic liver ( G , H ) . Weak EN expression in portal tracts of normal liver ( E ) . Abundant EN expression detected in activated HSC in fibrous septa of fibrotic ( F ) , active cirrhotic ( G ) , and inactive cirrhotic liver ( H ) . Scale bars : 500 μm . I - N Higher magnification of EN - stained normal liver ( I ) , fibrotic liver ( K ) , and ( active ) cirrhotic liver ( M ) compared to alpha - smooth muscle actin ( αSMA ) immunohistochemistry on serial slides ( J , L and N ) . Scale bars : 100 μm . O EN / αSMA mRNA ratio in normal liver , fibrotic liver , and active cirrhotic liver . P Collagen 1a ( Col1a ) expression in normal , fibrotic , and active cirrhotic liver determined by qRT - PCR .

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Figure 1A EN qRT-PCR of murine liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), hepatocytes (Hep), and HSC (n = 3 per cell type).B EN qRT-PCR of human hepatocytes (n = 1) and HSC cell line LX-2 (n = 3).C EN immunofluorescence staining of LX-2 cells. Scale bar: 25 μm.D EN qRT-PCR of normal liver (NL) and fibrotic liver (FL,n = 8 each), and active or inactive cirrhotic liver (CL+/−, n = 10/ n = 2).E-H EN immunohistochemistry staining of human normal liver (E), fibrotic liver (F), and cirrhotic liver (G, H). Weak EN expression in portal tracts of normal liver (E). Abundant EN expression detected in activated HSC in fibrous septa of fibrotic (F), active cirrhotic (G), and inactive cirrhotic liver (H). Scale bars: 500 μm.I-N Higher magnification of EN-stained normal liver (I), fibrotic liver (K), and (active) cirrhotic liver (M) compared to alpha-smooth muscle actin (αSMA) immunohistochemistry on serial slides (J, L and N). Scale bars: 100 μm.O EN/αSMA mRNA ratio in normal liver, fibrotic liver, and active cirrhotic liver.P Collagen 1a (Col1a) expression in normal, fibrotic, and active cirrhotic liver determined by qRT-PCR.


Figure 2A , B qRT - PCR for EN ( A ) and αSMA ( B ) of liver lysates from single high - dose CCl4 - or oil - treated wild - type ( WT ) mice ( n = 2 per time point ) . C Early activated and long - term - activated HSC isolated by differential density gradient centrifugation ( 8 . 26 % versus 11 . 5 % density layer ) . qRT - PCR for EN from early activated ( 8 . 26 % ) and long - term - activated HSC ( 11 . 5 % ) from 4 - week CCL4 - treated mice and vehicle control mice ( n = 3 - 4 per data point ) . D EN expression in liver lysates from 2 - , 4 - , and 6 - week CCl4 - treated mice ( n = 5 - 8 ) and vehicle control mice ( n = 3 ) determined by qRT - PCR . E - G METAVIR score of 2 - , 4 - , and 6 - week CCl4 - treated mice ( F - G ) Col1a and F4 / 80 immunofluorescence of 6 - week CCl4 - treated and vehicle control WT ( F ) and ENKO ( G ) mice . H Mean Col1a + area per high power field ( HPF ) shown as percentage of the total area ( n = 5 - 8 ) . I Western blot analysis for collagen 1a ( Col1a ) , platelet - derived growth factor receptor beta ( PDGFRβ ) and β - actin of total liver protein lysates from 6 - week CCl4 - treated or control vehicle WT and ENKO mice ( n = 3 - 5 ) . J - L Double immunofluorescence of 6 - week CCl4 - treated and vehicle control WT ( J ) and ENKO ( K ) mice for desmin and αSMA ( L ) shown as percentage of the total area ( n = 5 - 8 ) . M Serum liver enzymes ( GPT / ALT and GOT / AST ) from 2 - , 4 - , and 6 - week CCl4 - treated WT and ENKO mice ( n = 5 - 8 ) . N qRT - PCR of endosialin ( EN ) , αSMA , and collagen 1a1 / 6a1 from lentiviral - mediated endosialin knockdown ( shEN ) and control - transfected ( CTL ) human hepatic stellate cells ( LX - 2 ) . O Lentiviral - mediated endosialin knockdown ( shEN ) and control ( CTL ) in LX - 2 cells ( n = 3 ) . Magnification 10 × .

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Figure 2A, B qRT-PCR for EN (A) and αSMA (B) of liver lysates from single high-dose CCl4- or oil-treated wild-type (WT) mice (n = 2 per time point).C Early activated and long-term-activated HSC isolated by differential density gradient centrifugation (8.26% versus 11.5% density layer). qRT-PCR for EN from early activated (8.26%) and long-term-activated HSC (11.5%) from 4-week CCL4-treated mice and vehicle control mice (n = 3-4 per data point).D EN expression in liver lysates from 2-, 4-, and 6-week CCl4-treated mice (n = 5-8) and vehicle control mice (n = 3) determined by qRT-PCR.E-G METAVIR score of 2-, 4-, and 6-week CCl4-treated mice (F-G) Col1a and F4/80 immunofluorescence of 6-week CCl4-treated and vehicle control WT (F) and ENKO (G) mice.H Mean Col1a+ area per high power field (HPF) shown as percentage of the total area (n = 5-8).I Western blot analysis for collagen 1a (Col1a), platelet-derived growth factor receptor beta (PDGFRβ) and β-actin of total liver protein lysates from 6-week CCl4-treated or control vehicle WT and ENKO mice (n = 3-5).J-L Double immunofluorescence of 6-week CCl4-treated and vehicle control WT (J) and ENKO (K) mice for desmin and αSMA (L) shown as percentage of the total area (n = 5-8).M Serum liver enzymes (GPT/ALT and GOT/AST) from 2-, 4-, and 6-week CCl4-treated WT and ENKO mice (n = 5-8).N qRT-PCR of endosialin (EN), αSMA, and collagen 1a1/6a1 from lentiviral-mediated endosialin knockdown (shEN) and control-transfected (CTL) human hepatic stellate cells (LX-2).O Lentiviral-mediated endosialin knockdown (shEN) and control (CTL) in LX-2 cells (n = 3). Magnification 10×.


Figure 3A Representative images of Ki - 67 immunohistochemistry after 2 , 4 , and 6 weeks of CCl4 treatment of WT and ENKO mice ( n = 5 - 8 mice per group and time point ) . Scale bars : 75 μm . B Quantitation of Ki67 + hepatocytes / HPF after 2 , 4 , and 6 weeks of CCl4 treatment . C Time course of EN expression after partial hepatectomy ( 2 / 3 PHx ) determined by qRT - PCR of total liver lysates ( n = 5 / time point , sham : n = 3 ) . D Representative images of Ki - 67immunohistochemistry 2 days after 2 / 3 PHx of WT and ENKOmice ( n = 5 - 9 mice per group and time point ) . E Quantitation of Ki - 67 + hepatocytes / HPF determined 1 , 2 , 4 , and 8 days after 2 / 3 PHx . F Number of mitotic figures 2 days after 2 / 3 PHx of WT and ENKOmice ( n = 5 - 9 mice per group and time point ) . G qRT - PCR analysis of total liver lysates 1 day after PHx for IGF2 . H Western blot analysis of phosphorylated insulin - like growth factor receptor 1 ( pIGFR1 ) , phosphorylated insulin receptor substrate 1 ( pIRS1 ) and β - actin 2 days after PHx . I Western blot analysis of phosphorylated and total extracellular signal - regulated kinase ( ERK ) and AKT 2 days after PHx . J , K qRT - PCR of IGF2 in isolated liver sinusoidal endothelial cells ( LSEC ) ( J ) or hepatocytes ( HEP ) ( K ) 1 day after PHx . L EdU - positive hepatocytes ( in % ) after 24 - h stimulation with conditioned medium ( CM ) from either WT or ENKO HSC ( 3 , 5 , or 7 days of activation ) . HGF ( 40 ng / ml ) and TGF - β ( 10 ng / ml ) serve as positive or negative control , respectively . M EdU - positive WT and ENKO hepatocytes ( in % ) after stimulation with IGF2 ( 100 ng / ml ) . HGF ( 40 or 10 ng / ml ) and TGF - β ( 10 ng / ml ) serve as positive or negative control , respectively .

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Figure 3A Representative images of Ki-67 immunohistochemistry after 2, 4, and 6 weeks of CCl4 treatment of WT and ENKO mice (n = 5-8 mice per group and time point). Scale bars: 75 μm.B Quantitation of Ki67+ hepatocytes/HPF after 2, 4, and 6 weeks of CCl4 treatment.C Time course of EN expression after partial hepatectomy (2/3 PHx) determined by qRT-PCR of total liver lysates (n = 5/time point, sham: n = 3).D Representative images of Ki-67immunohistochemistry 2 days after 2/3 PHx of WT and ENKOmice (n = 5-9 mice per group and time point).E Quantitation of Ki-67+hepatocytes/HPF determined 1, 2, 4, and 8 days after 2/3 PHx.F Number of mitotic figures 2 days after 2/3 PHx of WT and ENKOmice (n = 5-9 mice per group and time point).G qRT-PCR analysis of total liver lysates 1 day after PHx for IGF2.H Western blot analysis of phosphorylated insulin-like growth factor receptor 1 (pIGFR1), phosphorylated insulin receptor substrate 1 (pIRS1) and β-actin 2 days after PHx.I Western blot analysis of phosphorylated and total extracellular signal-regulated kinase (ERK) and AKT 2 days after PHx.J, K qRT-PCR of IGF2 in isolated liver sinusoidal endothelial cells (LSEC)(J) or hepatocytes (HEP)(K) 1 day after PHx.L EdU-positive hepatocytes (in %) after 24-h stimulation with conditioned medium (CM) from either WT or ENKO HSC (3, 5, or 7 days of activation). HGF (40 ng/ml) and TGF-β (10 ng/ml) serve as positive or negative control, respectively.M EdU-positive WT and ENKO hepatocytes (in %) after stimulation with IGF2 (100 ng/ml). HGF (40 or 10 ng/ml) and TGF-β (10 ng/ml) serve as positive or negative control, respectively.


Figure 1His - tagged GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD and SARS - CoV - 2 NTD proteins were incubated with BHK 21 cells expressing eGFP - fused ACE2s . Anti - His / APC antibody was used to detect cells expressing His - tagged protein . Cells stained with the GD / 1 / 2019 RBD , GX / P2V / 2017 , SARS - CoV - 2 RBD and SARS - CoV - 2 NTD proteins are shown in green , orange , marine and red , respectively . The SARS - CoV - 2 NTD was used as negative control .

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Figure 1His-tagged GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins were incubated with BHK 21 cells expressing eGFP-fused ACE2s. Anti-His/APC antibody was used to detect cells expressing His-tagged protein. Cells stained with the GD/1/2019 RBD, GX/P2V/2017, SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins are shown in green, orange, marine and red, respectively. The SARS-CoV-2 NTD was used as negative control.


Figure 2The mFc - tagged ACE2s were captured by anti - mIgG Fc antibodies immobilized on the CM5 chip , and sequentially tested the binding with serially diluted SARS - CoV - 2 RBD , GX / P2V / 2017 RBD and GD / 1 / 2019 RBD . The raw and fitted curves are displayed in dotted and solid lines , respectively . The binding affinity between the RBDs ( SARS - CoV - 2 RBD , GX / P2V / 2017 RBD and GD / 1 / 2019 RBD ) and the 13 ACE2s are shown . Mean ± SD represents the mean and standard deviation of three independent experiments .

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Figure 2The mFc-tagged ACE2s were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD. The raw and fitted curves are displayed in dotted and solid lines, respectively. The binding affinity between the RBDs (SARS-CoV-2 RBD, GX/P2V/2017 RBD and GD/1/2019 RBD) and the 13 ACE2s are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.


Figure 3Hela - hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS - CoV - 2 , GX / P2V / 2017 and GD / 1 / 2019 , respectively . Green fluorescence indicates Hela - hACE2 cells infected with pseudovirus . Untransfected HeLa cell is used as negative control . The scale bar indicates 400 μm . Statistic for the infectivity of the three pseudoviruses . Data represent the results of five replicates . All data are presented as mean ± SD . * * * p < 0 . 001 ( Student ' s t - test ) . ns , no significant differences .

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Figure 3Hela-hACE2 cells and untransfected HeLa cells are infected with the pseudotyped SARS-CoV-2, GX/P2V/2017 and GD/1/2019, respectively. Green fluorescence indicates Hela-hACE2 cells infected with pseudovirus. Untransfected HeLa cell is used as negative control. The scale bar indicates 400 μm. Statistic for the infectivity of the three pseudoviruses. Data represent the results of five replicates. All data are presented as mean ± SD. ***p < 0.001 (Student's t-test). ns, no significant differences.


Figure 6Flow cytometric assay of the SARS - CoV - 2 RBD and its mutants ( K417R , Q498H and K417R - Q498H ) binding to human , mouse , rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells . The SARS - CoV - 2 NTD was used as negative control . The mFc - tagged ACE2s from human , mouse , rat and European hedgehog were captured by anti - mIgG Fc antibodies immobilized on the CM5 chip , and sequentially tested the binding with serially diluted SARS - CoV - 2 RBD and its mutants ( K417R , Q498H and K417R - Q498H ) . The binding affinities of the four ACE2s to the SARS - CoV - 2 RBD and its mutants are shown . Mean ± SD represents the mean and standard deviation of three independent experiments .

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Figure 6Flow cytometric assay of the SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H) binding to human, mouse, rat and European hedgehog ACE2s expressed on the cell surface of BHK21 cells. The SARS-CoV-2 NTD was used as negative control.The mFc-tagged ACE2s from human, mouse, rat and European hedgehog were captured by anti-mIgG Fc antibodies immobilized on the CM5 chip, and sequentially tested the binding with serially diluted SARS-CoV-2 RBD and its mutants (K417R, Q498H and K417R-Q498H).The binding affinities of the four ACE2s to the SARS-CoV-2 RBD and its mutants are shown. Mean ± SD represents the mean and standard deviation of three independent experiments.


Figure 2A ) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions , as well as SCAN - B whole mRNA . B ) Number of non - synonymous mutations per sample . Bars are colored by PAM50 subtypes Luminal A ( dark blue ) , Luminal B ( light blue ) , HER2 - enriched ( pink ) , basal - like ( red ) , Normal - like ( green ) and Unclassified ( gray ) . C - F ) Lollipop plots showing the location , abundance , and impact of SNVs in C ) TP53 , D ) PIK3CA , E ) PTEN , and F ) ERBB2 on the respective encoded protein . Protein change labels are shown for the most mutated amino acid positions , with residues ordered left to right by mutation frequency within each label .

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Figure 2A) Contribution of base change types to the overall SNV composition in the ABiM cohort for captured DNA regions and mRNA in the captured DNA regions, as well as SCAN-B whole mRNA.B) Number of non-synonymous mutations per sample. Bars are colored by PAM50 subtypes Luminal A (dark blue), Luminal B (light blue), HER2-enriched (pink), basal-like (red), Normal-like (green) and Unclassified (gray). C-F) Lollipop plots showing the location, abundance, and impact of SNVs in C) TP53, D) PIK3CA, E) PTEN, and F) ERBB2 on the respective encoded protein. Protein change labels are shown for the most mutated amino acid positions, with residues ordered left to right by mutation frequency within each label.


Figure 3Waterfall plot of the 20 most frequently mutated genes ( rows ) across 3 , 217 SCAN - B samples ( columns ) . Genes are ranked from top to bottom by mutation frequency . Samples are sorted by histological subtype and alteration occurrence . Mutations are colored by predicted functional impact .

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Figure 3Waterfall plot of the 20 most frequently mutated genes (rows) across 3,217 SCAN-B samples (columns). Genes are ranked from top to bottom by mutation frequency. Samples are sorted by histological subtype and alteration occurrence. Mutations are colored by predicted functional impact.


Figure 5 A - E ) Overall survival ( OS ) of patients with tumors containing mutations in the genes A ) TP53 , B ) PIK3CA , C ) ERBB2 , and D ) PTEN . E ) OS by PTEN - MutExp genotype ( " low " defined as PTEN mutation or PTEN expression in the lower quartile across the cohort , " normal " otherwise ) stratified by groups receiving no systemic treatment ( n = 336 ) , endocrine therapy only ( Endo only ; n = 1 , 579 ) , endocrine and chemotherapy ( Endo + Chemo ± any ; n = 914 ) , as well as HER2 treatment with any other treatment or none ( HER2 ± any ; n = 348 ) . Specific treatments in these groups are detailed in Table EV5 . In each Kaplan - Meier plot , wildtype ( wt ) and normal cases are plotted in blue , mutated ( mut ) and low cases are plotted in red , the log - rank P value is given , and the hazard ratio ( HR ) for mutation / low is given with a 95 % CI and after uni - variable and multi - variable ( MV ) Cox regression adjustment . Covariables included in the MV analysis were age at diagnosis , lymph node status , tumor size , and the variables denoted by the following symbols : ¶ , ER , PgR , HER2 , and NHG ; ¤ , ER , PgR , and NHG ; $ , HER2 and NHG . Abbreviations : ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade .

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Figure 5 A-E) Overall survival (OS) of patients with tumors containing mutations in the genes A) TP53, B) PIK3CA, C) ERBB2, and D) PTEN. E) OS by PTEN-MutExp genotype ("low" defined as PTEN mutation or PTEN expression in the lower quartile across the cohort, "normal" otherwise) stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemotherapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV5. In each Kaplan-Meier plot, wildtype (wt) and normal cases are plotted in blue, mutated (mut) and low cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation/low is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.


Figure 6 A - E ) Overall survival of patients with tumors containing mutations in pathways A ) WNT signaling , B ) Hedgehog signaling , C ) NOTCH2 signaling , D ) p53 independent DNA damage repair , E ) TGFβ signaling , stratified by groups receiving no systemic treatment ( n = 336 ) , endocrine therapy only ( Endo only ; n = 1 , 579 ) , endocrine and chemo therapy ( Endo + Chemo ± any ; n = 914 ) , as well as HER2 treatment with any other treatment or none ( HER2 ± any ; n = 348 ) . Specific treatments in these groups are detailed in Table EV4 . In each Kaplan - Meier plot , wildtype ( wt ) cases are plotted in blue , mutated ( mut ) cases are plotted in red , the log - rank P value is given , and the hazard ratio ( HR ) for mutation is given with a 95 % CI and after uni - variable and multi - variable ( MV ) Cox regression adjustment . Covariables included in the MV analysis were age at diagnosis , lymph node status , tumor size , and the variables denoted by the following symbols : ¶ , ER , PgR , HER2 , and NHG ; ¤ , ER , PgR , and NHG ; $ , HER2 and NHG . Abbreviations : ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade .

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Figure 6 A-E) Overall survival of patients with tumors containing mutations in pathways A) WNT signaling, B) Hedgehog signaling, C) NOTCH2 signaling, D) p53 independent DNA damage repair, E) TGFβ signaling, stratified by groups receiving no systemic treatment (n=336), endocrine therapy only (Endo only; n=1,579), endocrine and chemo therapy (Endo + Chemo ± any; n=914), as well as HER2 treatment with any other treatment or none (HER2 ± any; n=348). Specific treatments in these groups are detailed in Table EV4. In each Kaplan-Meier plot, wildtype (wt) cases are plotted in blue, mutated (mut) cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for mutation is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG. Abbreviations: ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade.