( 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 .

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

(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 .

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

(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 .

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

(D) 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 .

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

(E) 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 .

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

(F) 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 .

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

(G) 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 .

(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 .

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

(I) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 ) .

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

(A-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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O

(Q, Western blot analysis of LATS1, LATS2, and YAP in (Q) C4-2 cells treated with the indicated siRNA.


( 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 ) .

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

(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 ) .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


( A ) Western blot analysis of AR ( left ) and quantification of AR ( right ) in C4 - 2 cells treated CHX for the indicated time .

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

(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 ) .

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

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

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.


relative 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 ) .

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

relative 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 ) .

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

(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 .

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

(H-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 .

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 - 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 ) .

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

(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).


( 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 .

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

(D) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-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).


Western 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 ) .

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

Western 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).


( 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 .

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

(A-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 .

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

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.


C ) 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 .

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

C) 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.


A ) 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 .

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

A) 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 ( ) .

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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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D, E) Kinetics of L-[3H]Leu (D) or L-[3H]Trp (E) uptake.


( 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 .

(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 ) .

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(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 .

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(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 .

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(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.


( 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 .

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(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 ) .

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(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 .

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(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 ) .

(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).


( B ) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP - COP1 constructs and untagged TRIB1 .

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(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 .

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(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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(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).


( 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 .

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(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 ) .

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(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 . .

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(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 .

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(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 ) .

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(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).


( 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 .

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(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 .

(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 ) .

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(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 .

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(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 ) .

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(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 .

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(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.


A EN qRT - PCR of murine liver sinusoidal endothelial cells ( LSEC ) , Kupffer cells ( KC ) , hepatocytes ( Hep ) , and HSC ( n = 3 per cell type ) .

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A 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 ) .

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 .

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 ) .

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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 .

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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 .

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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P Collagen 1a (Col1a) expression in normal, fibrotic, and active cirrhotic liver determined by qRT-PCR.


A , 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 ) .

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A, 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 ) .

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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 .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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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O Lentiviral-mediated endosialin knockdown (shEN) and control (CTL) in LX-2 cells (n = 3). Magnification 10×.


A 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 .

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A 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 ) .

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 ) .

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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 .

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 .

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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 .

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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 .

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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 .

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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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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.


His - 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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His-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.


The 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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The 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.


Hela - 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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Hela-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.


Flow 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 .

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Flow 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 ) .

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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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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.


A ) 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 .

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A) 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 ) .

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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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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.


Waterfall 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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Waterfall 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.


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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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.


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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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.


Overall survival stratified by tumor mutational burden ( TMB ) across treatment groups in 3 , 217 patients . Samples were classified as TMB - high if the amount of non - synonymous mutations per expressed MB ( rnaMB ) was ≥ the median number of non - synonymous mutations per rnaMB across the whole SCAN - B cohort ( 0 . 082 mutations per rnaMB ) and TMB - low otherwise . In each Kaplan - Meier plot , TMB - low cases are plotted in blue , TMB - high cases are plotted in red , the log - rank P value is given , and the hazard ratio ( HR ) for TMB - high 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 ; # , NHG . Abbreviations : TMB , tumor mutational burden ; ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade ; HoR , hormone receptor ; TNBC , triple - negative breast cancer .

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

Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high 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; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.


B RT - PCR on cytoplasmic ( C ) and nuclear ( N ) extracts showing the subcellular localization of lnc - SMaRT . RNA was isolated from C2C12 cells after two days of differentiation . GAPDH mRNA and pre - mRNA ( pre - GAPDH ) were used respectively as cytoplasmic and nuclear controls . Representative results from three independent experiments are shown .

B RT-PCR on cytoplasmic (C) and nuclear (N) extracts showing the subcellular localization of lnc-SMaRT. RNA was isolated from C2C12 cells after two days of differentiation. GAPDH mRNA and pre-mRNA (pre-GAPDH) were used respectively as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown.


C qPCR analysis of lnc - SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points . The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as the mean ± s . e . m . of three biological replicates ( dots ) .

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C qPCR analysis of lnc-SMaRT RNA expression in C2C12 cells undergoing differentiation at the indicated time points. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots).


D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs ( si - SCR ) or with two different siRNAs against lnc - SMaRT ( si - SMaRT - 1 , si - SMaRT - 2 ) and induced to differentiate for two days . The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1 . Data are presented as the mean ± s . e . m . of three biological replicates ( dots ) . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * * P < 0 . 001 .

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D qPCR on RNA extracts from C2C12 myoblasts treated with either control siRNAs (si-SCR) or with two different siRNAs against lnc-SMaRT (si-SMaRT-1, si-SMaRT-2) and induced to differentiate for two days. The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to GM samples set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


E Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI staining ( in blue ) C2C12 murine myoblasts transfected with either control ( si - SCR ) or lnc - SMaRT siRNAs ( si - SMaRT - 1 , si - SMaRT - 2 ) fixed after at two days of differentiation . Histograms represent MHC - positive mononucleated cells / total MHC - positive cells ratio and fusion index quantification ( F . I . ) . At least 5 randomly chosen microscope fields of two independent biological samples were analyzed ( n > 600 cells for each field ) . Data are presented as the mean . of the two biological replicates ( dots ) .

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E Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) C2C12 murine myoblasts transfected with either control (si-SCR) or lnc-SMaRT siRNAs (si-SMaRT-1, si-SMaRT-2) fixed after at two days of differentiation. Histograms represent MHC-positive mononucleated cells/total MHC-positive cells ratio and fusion index quantification (F.I.). At least 5 randomly chosen microscope fields of two independent biological samples were analyzed (n>600 cells for each field). Data are presented as the mean. of the two biological replicates (dots).


A Gene expression heatmap showing the hierarchical clustering of protein - coding genes whose expression is altered upon lnc - SMaRT depletion . Expression values , calculated as RPKMs , were log2 - transformed and mean centered . Only genes with an average RPKM between 10 and 1000 were plotted .

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A Gene expression heatmap showing the hierarchical clustering of protein-coding genes whose expression is altered upon lnc-SMaRT depletion. Expression values, calculated as RPKMs, were log2-transformed and mean centered. Only genes with an average RPKM between 10 and 1000 were plotted.


B Volcano plot describing the differential gene expression between si - SCR and si - SMaRT treated C2C12 samples . For each gene , log2 of fold - change versus - log10 of the unadjusted p - value is plotted . Points in red represent differentially expressed genes ( FDR adjusted p - value < 0 . 05 ) .

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B Volcano plot describing the differential gene expression between si-SCR and si-SMaRT treated C2C12 samples. For each gene, log2 of fold-change versus -log10 of the unadjusted p-value is plotted. Points in red represent differentially expressed genes (FDR adjusted p-value < 0.05).


D Western blot on protein extract from control and lnc - SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation . CASP3 antibody was used . ACTN was used as loading control . Representative results from three independent experiments are shown .

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D Western blot on protein extract from control and lnc-SMaRT overexpressing C2C12 stable cell lines collected after one day of differentiation. CASP3 antibody was used. ACTN was used as loading control. Representative results from three independent experiments are shown.


A Localization on the lnc - SMaRT transcript of the two sets of biotinylated probes ( Set # 1 and Set # 2 ) used for lnc - SMaRT pull - down experiment .

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A Localization on the lnc-SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc-SMaRT pull-down experiment.


B Left panel : qPCR analysis of lnc - SMaRT enrichment in the RNA pull - down performed in C2C12 cells at day 2 of differentiation ( DM2 ) ; Set # 1 and Set # 2 probes were used against lnc - SMaRT together with a control set of probes against LacZ mRNA ( LacZ ) . Data are expressed in percentage of input and presented as the mean ± s . e . m . of three biological replicates ( dots ) . Right panel : Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc - SMaRT pull - down ; GAPDH was used as the negative control . Representative results from three independent experiments are shown .

B Left panel: qPCR analysis of lnc-SMaRT enrichment in the RNA pull-down performed in C2C12 cells at day 2 of differentiation (DM2); Set#1 and Set#2 probes were used against lnc-SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots). Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc-SMaRT pull-down; GAPDH was used as the negative control. Representative results from three independent experiments are shown.


C Upper panel : Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation . Input sample ( IN ) accounts for 2 . 5 % of the extract . Representative results from three independent experiments are shown . Lower panel : qPCR analysis of lnc - SMaRT enrichment in DHX36 RIP - derived RNA extracts . WBP4 was used as positive control [ 31 ] , while Neat1 ( a lncRNA expressed at comparable level of lnc - SMaRT according to the RNA - sequencing ) and Rps7 were used as negative controls . Data are expessed as percentage of input normalized on IgG control , and presented as the mean ± s . e . m . of three biological replicates ( dots ) .

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C Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown. Lower panel: qPCR analysis of lnc-SMaRT enrichment in DHX36 RIP-derived RNA extracts. WBP4 was used as positive control [31], while Neat1 (a lncRNA expressed at comparable level of lnc-SMaRT according to the RNA-sequencing) and Rps7 were used as negative controls. Data are expessed as percentage of input normalized on IgG control, and presented as the mean ± s.e.m. of three biological replicates (dots).


D RT - PCR validation of Mlx - α , Mlx - β and MLX - γ mRNA enrichment upon the lnc - SMaRT pull - down performed with Set # 1 and Set # 2 probes ; a control set of probes against LacZ mRNA ( LacZ ) was used as negative control . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown .

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D RT-PCR validation of Mlx-α, Mlx-β and MLX-γ mRNA enrichment upon the lnc-SMaRT pull-down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.


E RT - PCR analysis of Mlx - α , Mlx - β and Mlx - γ mRNA enrichment in DHX36 RIP - derived RNA extracts in samples treated with control siRNA ( siSCR , upper panel ) or siRNA against lnc - SMaRT ( siSMaRT , lower panel ) . GAPDH was used as negative control . Input sample ( IN ) accounts for 10 % of the extract . Representative results from three independent experiments are shown .

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E RT-PCR analysis of Mlx-α, Mlx-β and Mlx-γ mRNA enrichment in DHX36 RIP-derived RNA extracts in samples treated with control siRNA (siSCR, upper panel) or siRNA against lnc-SMaRT (siSMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown.


F Upper panel : Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG - tagged MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . FLAG - tag has been inserted at the N - terminus of MLX protein isoforms . HPRT was used as loading control . Representative results from three independent experiments are shown . Middle panel : Quantification of FLAG - tagged protein levels normalized on HPRT signals . Data are expressed presented as the mean ± s . d . of three biological replicates ( dots ) . Statistical analysis was performed with two - way ANOVA followed by Bonferroni ' s multiple comparisons test . * * * P < 0 . 001 . Lower panel : RT - PCR analysis of lnc - SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged - MLX ( MLX - α , MLX - β and MLX - γ ) in control condition ( CTRL ) or in overexpression of lncSMaRT ( OE - SMaRT ) . GAPDH was used as loading control . Representative results from three independent experiments are shown .

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F Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG-tagged MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). FLAG-tag has been inserted at the N-terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown. Middle panel: Quantification of FLAG-tagged protein levels normalized on HPRT signals. Data are expressed presented as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two-way ANOVA followed by Bonferroni's multiple comparisons test. ***P < 0.001. Lower panel: RT-PCR analysis of lnc-SMaRT and Mlx expression on RNA extracts from HeLa cells overexpressing the indicated isoforms of flagged-MLX (MLX-α, MLX-β and MLX-γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE-SMaRT). GAPDH was used as loading control. Representative results from three independent experiments are shown.


G Upper panel : Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector ( CTRL ) or with a plasmid overexpressing FLAG - tagged MLX - γ ( MLX - γ ) in control conditions ( si - SCR ) or in knockdown of DHX36 ( si - DHX36 ) . ACTININ was used as loading control . Representative results from three independent experiments are shown . Lower panel : RT - PCR analysis of Mlx - γ expression on the described RNA extracts . GAPDH was used as loading control . Representative results from three independent experiments are shown .

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G Upper panel: Western blot with FLAG antibodies on protein extracts from N2a cells transfected with an empty vector (CTRL) or with a plasmid overexpressing FLAG-tagged MLX-γ (MLX-γ) in control conditions (si-SCR) or in knockdown of DHX36 (si-DHX36). ACTININ was used as loading control. Representative results from three independent experiments are shown. Lower panel: RT-PCR analysis of Mlx-γ expression on the described RNA extracts. GAPDH was used as loading control. Representative results from three independent experiments are shown.


A Immunofluorescence for total MLX protein detection ( red signal ) performed on C2C12 cells treated with siRNAs against Mlx - γ ( si - Mlx - γ ) , lnc - SMaRT ( si - SMaRT ) , DHX36 ( si - DHX36 ) or with a control siRNA ( si - SCR ) . Cells were fixed two days after the switch to differentiation medium . Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment . The inserts on the topside of the images sketch the MLX signal peaks in the nuclei ( white stars ) to highlight the variation of MLX staining in each condition . Dashed lines indicate the edge of the nucleus .

A Immunofluorescence for total MLX protein detection (red signal) performed on C2C12 cells treated with siRNAs against Mlx-γ (si-Mlx-γ), lnc-SMaRT (si-SMaRT), DHX36 (si-DHX36) or with a control siRNA (si-SCR). Cells were fixed two days after the switch to differentiation medium. Representative binary images of single confocal planes for MLX immunofluorescence are shown for each treatment. The inserts on the topside of the images sketch the MLX signal peaks in the nuclei (white stars) to highlight the variation of MLX staining in each condition. Dashed lines indicate the edge of the nucleus.


B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear ( Nuc ) and cytoplasmic ( Cyt ) compartment in the indicated conditions . About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates ( dots ) .

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B Histogram represents the fluorescence intensity ratio of the MLX protein signals in the nuclear (Nuc) and cytoplasmic (Cyt) compartment in the indicated conditions. About 50 cells from two independent experiments were analyzed Data are presented as the mean of two biological replicates (dots).


A Left panel : schematic representation of the luciferase constructs produced for Mlx . Mlx 5 ' - and 3 ' UTR - interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region ( RLuc - Mlx 5 ' - 3 ' ) and mutant derivatives devoid of the 5 ' interacting region ( RLuc - Mlx - 3 ' ) or the 3 ' UTR interacting region ( RLuc - Mlx - 5 ' ) were obtained . These constructs were co - transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx 5 ' - 3 ' , RLuc - Mlx - 3 ' , RLuc - Mlx - 5 ' ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * P < 0 . 05 .

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A Left panel: schematic representation of the luciferase constructs produced for Mlx. Mlx 5'- and 3'UTR-interacting regions were cloned respectively upstream and downstream the Renilla luciferase coding region (RLuc-Mlx 5'-3') and mutant derivatives devoid of the 5' interacting region (RLuc-Mlx-3') or the 3' UTR interacting region (RLuc-Mlx-5') were obtained. These constructs were co-transfected in C2C12 myoblasts in growth conditions together with plasmids expressing lnc-SMaRT (+) or with a control vector (-). Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx 5'-3', RLuc-Mlx-3', RLuc-Mlx-5') set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. *P < 0.05.


B The RLuc - Mlx - 5 ' construct was co - transfected in N2a cells together with plasmids expressing lnc - SMaRT ( + ) or with a control vector ( - ) and treated with control siRNA ( + ) or siRNA against DHX36 ( - ) . Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to RLuc - Mlx - 5 ' vector set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * P < 0 . 01 , * * * P < 0 . 001 .

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B The RLuc-Mlx-5' construct was co-transfected in N2a cells together with plasmids expressing lnc-SMaRT (+) or with a control vector (-) and treated with control siRNA (+) or siRNA against DHX36 (-). Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to RLuc-Mlx-5' vector set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. **P < 0.01, ***P < 0.001.


C Left panel : schematic representation of the luciferase constructs used . The RLuc - Mlx - 5 ' construct and its derived mutants ( RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) were transfected in N2a cells in growth conditions together with lnc - SMaRT - expressing plasmids ( lnc - SMaRT ) or with its derived mutant ( lnc - SMaRTmut ) in the indicated combinations . Right panel : Luciferase activity data , presented as the mean ± s . e . m . of three biological replicates ( dots ) , are shown with respect to each RLuc control vector ( RLuc - Mlx - 5 ' , RLuc - Mlxmut30 , RLuc - Mlx Δ75 ) set to a value of 1 . Statistical analysis was performed with ordinary analysis of variance ( ANOVA ) followed by Dunnett ' s multiple comparison test . * * * P < 0 . 001 .

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C Left panel: schematic representation of the luciferase constructs used. The RLuc-Mlx-5' construct and its derived mutants (RLuc-Mlxmut30, RLuc-Mlx Δ75) were transfected in N2a cells in growth conditions together with lnc-SMaRT-expressing plasmids (lnc-SMaRT) or with its derived mutant (lnc-SMaRTmut) in the indicated combinations. Right panel: Luciferase activity data, presented as the mean ± s.e.m. of three biological replicates (dots), are shown with respect to each RLuc control vector (RLuc-Mlx-5', RLuc-Mlxmut30, RLuc-Mlx Δ75) set to a value of 1. Statistical analysis was performed with ordinary analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. ***P < 0.001.


A Upper panel : schematic representation of the predicted base pairing between lnc - SMaRT ( region A ) and Mlx γ mRNA ; the predicted G - 4 structure is boxed in red while the synthetic oligos sequences are underlined in red . Lower panel : Lane 1 and 2 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA ( SYBR GOLD , indicated as SYBR in the figure ) . Lane 3 and 4 : 5 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 5 and 6 : 200 pmol of lnc - SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX ( NMM ) . Lane 7 and 8 : 5 pmol of Mlx oligo alone ( 7 ) or in combination with 5pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained for total RNA ( SYBR GOLD ) . Lane 9 and 10 : 200 pmol of Mlx oligo alone ( 7 ) or in combination with 200 pmol of lnc - SMaRT oligo ( 8 ) were heated at 100 ° C for 10 min and , after slow cooling , loaded on a native polyacrylamide gel and stained with the selective G - quadruplex staining N - methyl mesoporphyrin IX . Asterisk ( * ) indicates the second NMM - positive band obtained upon SMaRT and γ - oligo interaction . Data information : Representative gels from at least three independent experiments are shown .

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A Upper panel: schematic representation of the predicted base pairing between lnc-SMaRT (region A) and Mlx γ mRNA; the predicted G-4 structure is boxed in red while the synthetic oligos sequences are underlined in red. Lower panel: Lane 1 and 2: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a denaturing polyacrylamide gel and stained for total RNA (SYBR GOLD, indicated as SYBR in the figure). Lane 3 and 4: 5 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 5 and 6: 200 pmol of lnc-SMaRT and Mlx synthetic oligos were loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX (NMM). Lane 7 and 8: 5 pmol of Mlx oligo alone (7) or in combination with 5pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained for total RNA (SYBR GOLD). Lane 9 and 10: 200 pmol of Mlx oligo alone (7) or in combination with 200 pmol of lnc-SMaRT oligo (8) were heated at 100°C for 10 min and, after slow cooling, loaded on a native polyacrylamide gel and stained with the selective G-quadruplex staining N-methyl mesoporphyrin IX. Asterisk (*) indicates the second NMM-positive band obtained upon SMaRT and γ-oligo interaction. Data information: Representative gels from at least three independent experiments are shown.