Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for NDP52 , capsid or actin ( M ) . Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean + s . d . ( n = 3 ) . Scale bar , 10 μm . C , capsid ; CHIKV , Chikungunya virus ; CTRL , control ; NDP52 , nuclear dot protein 52 kDa ; qRT ‐ PCR , quantitative reverse transcription polymerase chain reaction ; siRNA , short interfering RNA .

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Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. C, capsid; CHIKV, Chikungunya virus; CTRL, control; NDP52, nuclear dot protein 52 kDa; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; siRNA, short interfering RNA.


p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells . Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2 , capsid or nonspecific IgG controls . Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid ( A , B ) .

B-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O

p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells. Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2, capsid or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid (A,B).


Cells were transfected with Ub ‐ HA and capsid ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . using antibodies to FLAG or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ HA antibody ( C ) .

O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O

Cells were transfected with Ub‐HA and capsid‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐HA antibody (C).


Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls . Immunoprecipitated proteins were revealed using antibodies to p62 , FK2 or capsid ( D , E ) .

B-EXP_ASSAY O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O

Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to p62, FK2 or capsid (D,E).


Cells were infected for 3 h and then transfected with empty plasmid ( CTRL ) or p62WT ‐ 3XFLAG or p62deltaUBA ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . with anti ‐ FLAG antibody . Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid ( F ) .

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Cells were infected for 3 h and then transfected with empty plasmid (CTRL) or p62WT‐3XFLAG or p62deltaUBA‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. with anti‐FLAG antibody. Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid (F).


Cells were treated with CTRL or p62 siRNA , then infected for 24 h . Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls . Immunoprecipitated proteins were revealed using anti ‐ capsid antibody ( G ) .

O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O

Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using anti‐capsid antibody (G).


Cells were infected for 15 h and labeled with antibodies to p62 , LAMP ‐ 1 or capsid ( H ) .

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Cells were infected for 15 h and labeled with antibodies to p62, LAMP‐1 or capsid (H).


Cells were infected for 9 , 15 or 24 h and treated with DMSO ( CTRL ) or bafilomycin 3 h before the end of experiment . Whole ‐ cell lysates were immunoblotted for capsid or actin ( I ) .

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Cells were infected for 9, 15 or 24 h and treated with DMSO (CTRL) or bafilomycin 3 h before the end of experiment. Whole‐cell lysates were immunoblotted for capsid or actin (I).


HeLa cells were transfected with empty plasmid ( CTRL ) , control protein ( cherry ‐ 3XFLAG ) or capsid ‐ 3XFLAG at fixed ( 750 ng ) or at the indicated concentrations . Cell mortality ( that is , % of dead cells ) was measured 24 h post transfection ( J , K ) .

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HeLa cells were transfected with empty plasmid (CTRL), control protein (cherry‐3XFLAG) or capsid‐3XFLAG at fixed (750 ng) or at the indicated concentrations. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (J,K).


Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 48 h post transfection ( L ) .

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Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (L).


Cells were infected with CHIKV for 3 h and transfected with empty plasmid ( CTRL ) , p62WT ‐ 3XFLAG , p62deltaUBA ‐ 3XFLAG or p62deltaLIR ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 24 h post transfection ( M ) .

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Cells were infected with CHIKV for 3 h and transfected with empty plasmid (CTRL), p62WT‐3XFLAG, p62deltaUBA‐3XFLAG or p62deltaLIR‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 24 h post transfection (M).


Human NDP52 binds nsP2 , localizes near CHIKV RCs , and restricts cell shutoff promoting cell survival in HeLa cells . Yeast cells expressing Gal4 DNA BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants ( top ) . Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone ( empty vector ) or to NDP52 ( bottom ) ( A ) .

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Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants (top). Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone (empty vector) or to NDP52 (bottom) (A).


Cells were infected for 15 h , then labeled using antibodies to NDP52 and nsP2 ( B ) , or to TGN46 , nsP2 and nsP3 ( C ) .

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Cells were infected for 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C).


Cells were treated with control ( CTRL ) ( left ) or NDP52 ( right ) siRNA , infected for 15 h , then labeled using antibodies to NDP52 , nsP2 and puromycin . Quantitative analysis was performed by counting the percentage of infected cells with TGN ‐ associated RCs ( n = 30 cells per experiment ) ( D ) .

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Cells were treated with control (CTRL) (left) or NDP52 (right) siRNA, infected for 15 h, then labeled using antibodies to NDP52, nsP2 and puromycin. Quantitative analysis was performed by counting the percentage of infected cells with TGN‐associated RCs (n=30 cells per experiment) (D).


Cells were transfected with empty plasmid ( CTRL ) or nsP2wt ‐ 3XFLAG or nsP2R606A ‐ 3XFLAG . Cell mortality ( that is , % of dead cells ) was measured 24 h post transfection ( E ) .

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Cells were transfected with empty plasmid (CTRL) or nsP2wt‐3XFLAG or nsP2R606A‐3XFLAG. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (E).


Cells were treated with control ( CTRL ) or NDP52 siRNA , then mock infected ( M ) or infected ( C ) for 24 h . Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52 . The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 ( F ) .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O

Cells were treated with control (CTRL) or NDP52 siRNA, then mock infected (M) or infected (C) for 24 h. Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52. The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 (F).


Cells were treated with control ( CTRL ) or NDP52 siRNA , infected for 15 and 24 h and treated with puromycin . Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for NDP52 , capsid or actin . Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin ( G ) .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O

Cells were treated with control (CTRL) or NDP52 siRNA, infected for 15 and 24 h and treated with puromycin. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin. Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin (G).


Cells were treated with CTRL or NDP52 siRNA , then transfected with NT or nsP2 ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 48 h post transfection ( H ) .

O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cells were treated with CTRL or NDP52 siRNA, then transfected with NT or nsP2‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (H).


NDP52 promotes CHIKV infection in a species ‐ specific manner . MEF cells were treated with CTRL or NDP52 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( A ) and viral production ( B ) were measured . Yeast cells expressing Gal4 DNA BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 ( C ) .

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NDP52 promotes CHIKV infection in a species‐specific manner. MEF cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (A) and viral production (B) were measured. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 (C).


MEF cells were infected for 3 h and transfected with empty plasmid ( CTRL ) or hNDP52 ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . using antibodies to FLAG or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ nsP2 antibody ( D ) .

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MEF cells were infected for 3 h and transfected with empty plasmid (CTRL) or hNDP52‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐nsP2 antibody (D).


MEF cells were infected for 3 h and transfected with empty plasmid ( CTRL ) , hNDP52 , hLC3 ‐ C or hNDP52 and hLC3 ‐ C . Viral production ( E ) and cell mortality ( F ) were assessed 24 h post transfection . Isogenic wt or Atg5 − / − MEFs were infected for 24 h and cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( G ) and viral production ( H ) were assessed . MEF cells were infected and immunoprecipitation experiments were performed 24 h p . i . using antibodies to p62 or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ capsid antibody ( I ) .

B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-CELL_LINE O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O

MEF cells were infected for 3 h and transfected with empty plasmid (CTRL), hNDP52, hLC3‐C or hNDP52 and hLC3‐C. Viral production (E) and cell mortality (F) were assessed 24 h post transfection. Isogenic wt or Atg5−/− MEFs were infected for 24 h and cell mortality (that is, fold change relative to mock‐infected cells) (G) and viral production (H) were assessed. MEF cells were infected and immunoprecipitation experiments were performed 24 h p.i. using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐capsid antibody (I).


MEF cells were treated with CTRL or p62 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) and viral production were assessed . Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for p62 or actin ( K ) .

B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O

MEF cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J) and viral production were assessed. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for p62 or actin (K).


A . Endogenous Cyclin B1 - YFP localisation during mitosis live in RPE1 cells ( still from Movie EV1 ) .

O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O O O O O O

A. Endogenous Cyclin B1-YFP localisation during mitosis live in RPE1 cells (still from Movie EV1).


Immunofluorescence images ( B ) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O

Immunofluorescence images (B) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC.


C . Immunofluorescence quantifications ( C ) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC . Each dot represents a kinetochore , data is from 40 kinetochore pairs ( 13 cells , max 5 kinetochore pairs / cell ) .

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C. Immunofluorescence quantifications (C) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. Each dot represents a kinetochore, data is from 40 kinetochore pairs (13 cells, max 5 kinetochore pairs/cell).


D , E . Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole - arrested cells ( noco ) treated with the MPS1 inhibitors , AZ - 3146 ( 5μM ) or Reversine ( 500nM ) , either before ( D ) or after ( E ) mitotic entry .

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D,E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5μM) or Reversine (500nM), either before (D) or after (E) mitotic entry.


F . Immunofluorescence images of LacI - MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome

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F. Immunofluorescence images of LacI-MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1


G . Live imaging of Cyclin B1 - mCherry ( CycB1 - mCh ) in LacO - U2OS cells transfected with LacI - MAD1 - FL ( full length : aa1 - 718 ) or various LacI - MAD1 truncations ( amino acid numbers indicated ) .

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G. Live imaging of Cyclin B1-mCherry (CycB1-mCh) in LacO-U2OS cells transfected with LacI-MAD1-FL (full length: aa1-718) or various LacI-MAD1 truncations (amino acid numbers indicated).


H , I . Immunofluorescence images ( H ) and quantifications ( I ) of Cyclin B1 and MAD1 kinetochore levels in control ( MAD1 - WT ) or MAD1β HeLa cells ( two independent clones : C13 or C24 ) treated with nocodazole .

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H,I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 or C24) treated with nocodazole.


J , K . Immunofluorescence images ( J ) and quantification ( K ) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline - inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole . Cells were selected that had full MAD1 knockout in the doxycycline treatment ( this constituted approximately 30 % of cells ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells).


L . Relative kinetochore volumes occupied by Cyclin B1 and MAD1 ( relative to CenpC ) in nocodazole - arrested MAD1α and MAD1β cells ( calculated from experiments shown in ( H , I ) .

O O O B-SUBCELLULAR B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).


A . Elution profiles and SDS - PAGE for SEC runs on the indicated column of the Cyclin B1 : CDK1 complex ( blue profile ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . Note that the same Cyclin B1 : CDK1 elution profile and SDS - PAGE is also displayed as reference in panels D and F to improve clarity . For the same reason , the MBP - MAD1 : MAD2 elution profile and SDS - PAGE is also displayed in panels 2A - C . B . Elution profiles and SDS - PAGE for SEC runs on the indicated column of CDK1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . C . Elution profiles and SDS - PAGE for SEC runs on the indicated column of Cyclin B1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . D . Elution profiles and SDS - PAGE for SEC runs of the Cyclin B1 : CDK1 complex ( blue ) , MBP - MAD1 ∆ 93 : MAD2 ( green ) , and their combination ( red ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O

A. Elution profiles and SDS-PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red), and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS-PAGE is also displayed as reference in panels D and F to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS-PAGE is also displayed in panels 2A-C. B. Elution profiles and SDS-PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). C. Elution profiles and SDS-PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). D. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green), and their combination (red).


F . Elution profiles and SDS - PAGE for SEC runs of Cyclin B1 : CDK1 ( blue ) , MBP - MAD13EK : MAD2 ( E52K , E53K , and E56K mutations ; red ) , and their combination ( green ) . In A - D , individual potential binding partners were combined at a concentration of 5 µM .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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. Elution profiles and SDS-PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K, and E56K mutations; red), and their combination (green). In A-D, individual potential binding partners were combined at a concentration of 5 µM.


A . Western blot analysis of indicated vsv - MAD1 - WT or 3EK HeLa clones treated with or without doxycycline for 10 days .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O B-GENEPROD O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O

A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days.


B . Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole - arrested MAD1 - WT - C13 AND 3EK - C14 just after nuclear envelope breakdown ( early prometaphase ) or later in mitosis when the chromatin is condensed ( late prometaphase ) . Note , that early and late prometaphase was defined based on the level of chromatin condensation .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O

B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation.


C , D . Relative kinetochore volumes occupied by MAD1 ( C ) and ZW10 ( D ) ( relative to CenpC ) in MAD1 - WT and - 3EK cells in early and late prometaphase .

O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O

C,D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CenpC) in MAD1-WT and -3EK cells in early and late prometaphase.


E . Quantification of MAD1 kinetochore intensity from indicated MAD1 - WT and 3EK clones treated as in ( B ) .

O O O O B-GENEPROD B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O

E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B).


A - C . Immunofluorescence images ( A ) and quantifications ( B , C ) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with / without AZ - 3146 for 30 min to inhibit MPS1 . In all kinetochore intensity graphs , each dot represents a cell , horizontal lines indicate the median and vertical bars show the 95 % confidence interval . Note , when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole , which are normalised to 1 . The mean level of the normalised controls is indicated by the dotted lines . 30 cells from 3 experiments . Scale bars = 5µM . D . Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1 . 25 µM AZ - 3146 . Graph shows cumulative mean ( ± SEM ) of 3 experiments , 50 cells per condition per experiment .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM. D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


A . Immunofluorescence images of MAD1 and MAD1 - pT716 kinetochore levels in nocodazole - arrested RPE1 cells treated with or without AZ - 3146 . Scale bars = 5µM .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM.


B . Electron micrographs of rotary - shadowed MAD193 - 718 : MAD2 ( top row ) , MBP - MAD1 : MAD2 ( middle row ) , and MBP - MAD1 : MAD2 in complex with GST - CDK1 : Cyclin B1 ( bottom row ) . Scale bar = 50 nm .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O

B. Electron micrographs of rotary-shadowed MAD193-718:MAD2 (top row), MBP-MAD1:MAD2 (middle row), and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm.


C . Quantifications ( top ) and corresponding immunofluorescence images ( underneath ) of relative kinetochore MAD1 and MAD1 - pT716 levels in nocodazole - arrested of MAD1 - WT - C14 and MAD1 - 3EK - C12 cells treated with different doses of AZ - 3146 for 30 min . MG132 was included at the time of AZ - 3146 addition to prevent mitotic exit . Each dot represents a cell , horizontal lines indicate the median and error bars show the 95 % confidence interval . Note , when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . 30 cells from 3 experiments . Both kinetochore intensity graphs display data that is relative to the WT - C14 untreated controls , which are normalised to 1 . The mean level of the normalised controls is indicated by the dotted lines .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O 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. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.


D . Duration of mitotic arrest in MAD1 - WT C14 or MAD1 - 3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ - 3146 . Note , the 1 . 25 µM AZ - 3146 data is displayed in Figure 4D , but also included here to allow comparison with other drug doses . Graph shows cumulative mean ( ± SEM ) of 3 experiments , 50 cells per condition per experiment .

O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O 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. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


A . Representative images of immunofluorescence of α - smooth muscle actin ( α - SMA ) , pan Cytokeratin , Pan - hMENA , hMENA11a and E - cadherin expression in CAFs and autologous cancer cells ( Ep - PDAC ) obtained from enzymatically digested primary PDAC tissue of patient # 36 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 20 μm .

O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-DISEASE O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

A. Representative images of immunofluorescence of α-smooth muscle actin (α-SMA), pan Cytokeratin, Pan-hMENA, hMENA11a and E-cadherin expression in CAFs and autologous cancer cells (Ep-PDAC) obtained from enzymatically digested primary PDAC tissue of patient #36. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm.


B . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression levels ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) in normal fibroblasts derived from transplant donor ( P - NF ) , pancreatic serous cystadenoma # 71 and cancer associated fibroblasts ( n = 10 ) obtained from primary PDAC tissues . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression . Quantification of P # 110 is relative to the sample shown in the WB on the left .

O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

B. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal fibroblasts derived from transplant donor (P-NF), pancreatic serous cystadenoma #71 and cancer associated fibroblasts (n=10) obtained from primary PDAC tissues. Densitometry quantified data (bottom) of hMENA∆v6 expression. Quantification of P#110 is relative to the sample shown in the WB on the left.


C . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression levels ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) in normal lung fibroblasts ( L - NF ) , cancer associated fibroblasts obtained from NSCLC tissues ( n = 4 ) and normal dermal fibroblasts ( HNF ) . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression .

O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-TISSUE B-CELL_TYPE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-DISEASE O O O O O O O O B-TISSUE B-CELL_TYPE O B-CELL_TYPE O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-EXP_ASSAY O

C. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal lung fibroblasts (L-NF), cancer associated fibroblasts obtained from NSCLC tissues (n=4) and normal dermal fibroblasts (HNF). Densitometry quantified data (bottom) of hMENA∆v6 expression.


A . Representative images of immunofluorescence of Pan - hMENA ( yellow ) and α - SMA ( red ) in the primary PDAC tissue of patient # 138 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . The inset of the dashed area is provided on the right as a zoomed - in and cropped fluorescence image . Scale bar : 20 μm . αSMA ‑ positive CAFs are also positive for Pan - hMENA ( arrow ) .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-DISEASE O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O

A. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA‑positive CAFs are also positive for Pan-hMENA (arrow).


B . Representative images of immunofluorescence of Pan - hMENA ( yellow ) and α - SMA ( red ) in NSCLC case # 484 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . The inset of the dashed area is provided on the right as a zoomed - in and cropped fluorescence image . Scale bar : 20 μm . As in A , α - SMA signal is evident in stromal cells which are also positive for Pan - hMENA ( arrow ) .

O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O

B. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow).


C . Boxplots showing the mean mRNA expression of ENAH gene ( hMENA ) for 22 patients in the 10 groups of cell types ( Peng et al . , 2019 ) ( total number of cells : 38487 , mean of cells for patient : 1603 ) . Shown in each boxplot are the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) . Expressions from each cell - type group were compared to all other groups by using Mann - Whitney U - test ( two - sided ) and P - values were adjusted for multiple testing using the Benjamini - Hochberg method . Stromal cell - type groups with significantly up - regulated ENAH expression respect to other stromal groups are : Fibroblast , * * * q = 0 . 0002 ; Stellate , * * * q = 1 . 5e - 07 . Non - stromal cell - type groups with significantly up - regulated ENAH expression with respect to other non - stromal groups are : Ductal 1 , * * q = 0 . 0013 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-CELL_TYPE O O O B-CELL_TYPE O 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-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O

C. Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al., 2019) (total number of cells: 38487, mean of cells for patient: 1603). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q=0.0002; Stellate, ***q= 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q=0.0013.


D . Boxplots showing the mRNA expression of ENAH gene ( hMENA ) in the 7 groups of cell types from ( Lambrechts et al . , 2018 ) ( total n = 52 ) . Shown in each boxplot are the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) . Cell expression from each group were compared to all other stromal / not - stromal cells by using Mann - Whitney U - test ( two - sided ) and P - values were adjusted for multiple testing using the Benjamini - Hochberg method ( Fibroblast group vs other stromal groups , * * q = 0 . 0022 ; Other comparisons , q > 0 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O 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-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

D. Boxplots showing the mRNA expression of ENAH gene (hMENA) in the 7 groups of cell types from (Lambrechts et al., 2018) (total n=52). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Cell expression from each group were compared to all other stromal/not-stromal cells by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method (Fibroblast group vs other stromal groups, **q=0.0022; Other comparisons, q>0.05


A . Quantification of in vitro matrigel invasion assay ( bottom ) of P - CAF and L - CAF ( P - CAF # 36 , 138 and L - CAF # 189 , 484 ) transfected with control siRNA ( CNT ) or hMENA siRNA ( hMENA ( t ) ) indicating that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 reduces the invasive ability of CAFs with respect to siCNT CAFs . Number of invading cells was measured by counting 6 random fields . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Immunoblot showing hMENA / hMENAΔv6 expression ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) of the CAFs employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay (bottom) of P-CAF and L-CAF (P-CAF # 36, 138 and L-CAF #189, 484) transfected with control siRNA (CNT) or hMENA siRNA (hMENA(t)) indicating that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 reduces the invasive ability of CAFs with respect to siCNT CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot showing hMENA/hMENAΔv6 expression (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) of the CAFs employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.


B . Quantification of in vitro matrigel invasion assay ( bottom ) of P - NF and L - NF and P - CAF # 110 and L - CAF # 400 transfected with control or hMENA ∆ v6 expressing vectors , demonstrating that the overexpression of hMENA ∆ v6 isoform induced the invasiveness of P - NFs and L - NFs and / or P - and L - CAFs . Number of invading cells was measured by counting 6 random fields . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Immunoblot of hMENAΔv6 expression ( detected by the specific anti - hMENAΔv6 antibody ) in fibroblasts employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay (bottom) of P-NF and L-NF and P-CAF#110 and L-CAF#400 transfected with control or hMENA∆v6 expressing vectors, demonstrating that the overexpression of hMENA∆v6 isoform induced the invasiveness of P-NFs and L-NFs and/or P-and L-CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot of hMENAΔv6 expression (detected by the specific anti-hMENAΔv6 antibody) in fibroblasts employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.


A . Quantification of in vitro matrigel invasion assay of PANC - 1 cells cultured for 48 h with conditioned media ( CM ) of NFs ( P - NFs - CM ) , CAF low # 44 and # 110 and CAFs high # 36 and 138 . Histograms show the number of invading cells measured by counting 6 random fields . Data are presented as the mean ± SD of three biological replicates , performed at least in duplicate each . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay of PANC-1 cells cultured for 48 h with conditioned media (CM) of NFs (P-NFs-CM), CAF low #44 and #110 and CAFs high #36 and 138. Histograms show the number of invading cells measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates, performed at least in duplicate each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. * P <0.05, **P<0.01, ***P<0.001.


B . Quantification of in vitro matrigel invasion assay of PANC - 1 cultured for 48 h with CM derived from control P - CAFs # 36 ( siCNT - P - CAF - CM # 36 ) and hMENA / hMENA ∆ v6 silenced P - CAFs ( sihMENA ( t ) - P - CAF - CM # 36 ) , showing that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects PANC - 1 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . Cells invading matrigel were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O 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-GENEPROD O B-GENEPROD O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay of PANC-1 cultured for 48 h with CM derived from control P-CAFs#36 (siCNT-P-CAF-CM#36) and hMENA/hMENA∆v6 silenced P-CAFs (sihMENA(t)-P-CAF-CM#36), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects PANC-1 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.


C . Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media ( culture medium ) or conditioned media ( CM ) of L - CAF low # 400 and CAFs high # 189 , as described above . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media (culture medium) or conditioned media (CM) of L-CAF low #400 and CAFs high #189, as described above. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.


D . Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L - CAFs # 484 ( siCNT - L - CAF - CM # 484 ) and hMENA / hMENA ∆ v6 silenced L - CAFs ( sihMENA ( t ) - L - CAF - CM # 484 ) , showing that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects H1975 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . Cells invading matrigel were counted in 6 random fields . Data are presented as the mean ± SD of six replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O 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-GENEPROD O B-GENEPROD O O O O O O O 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-GENEPROD O B-GENEPROD O O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L-CAFs#484 (siCNT-L-CAF-CM#484) and hMENA/hMENA∆v6 silenced L-CAFs (sihMENA(t)-L-CAF-CM#484), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects H1975 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of six replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.


E . Representative immunoblot ( top ) and densitometry quantification ( bottom ) of hMENA ∆ v6 expression level in P - NFs grown in RPMI control medium ( - ) or PANC - 1 - CM for 24 h ( n = 3 ) . Data are represented as fold increase with respect to control medium ± SD ( set as 1 ) . Data were analyzed using two ‐ sided Student ' s t ‐ test . * P < 0 . 05 .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunoblot (top) and densitometry quantification (bottom) of hMENA∆v6 expression level in P-NFs grown in RPMI control medium (-) or PANC-1-CM for 24 h (n = 3). Data are represented as fold increase with respect to control medium ± SD (set as 1). Data were analyzed using two‐sided Student's t‐test. *P < 0.05.


F . Representative images ( top ) and quantification ( bottom ) of collagen gel - contraction ability of CAFs ( monoculture ) or CAFs co - cultured with siCNT ( co - siCNT ) or sihMENA ( t ) PANC - 1 cells ( co - sihMENA ( t ) ) . Dashed white circles illustrate the margins of the gel area . Data are presented as the mean ± SD of two biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 005 , followed by Bonferroni ' s multiple comparison test ANOVA : * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images (top) and quantification (bottom) of collagen gel-contraction ability of CAFs (monoculture) or CAFs co-cultured with siCNT (co-siCNT) or sihMENA(t) PANC-1 cells (co-sihMENA(t)). Dashed white circles illustrate the margins of the gel area. Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P=0.005, followed by Bonferroni's multiple comparison test ANOVA: *P<0.05, **P<0.01.


G . Representative immunoblot ( top ) and quantification ( bottom ) of hMENA ∆ v6 and α - SMA expression in P - CAFs # 110 treated with DMEM medium ( - ) or with conditioned medium ( CM ) derived from siCNT ( siCNT PANC - 1 ) or sihMENA ( t ) PANC - 1 cells ( ( sihMENA ( t ) PANC - 1 ) ( n = 3 ) . Data are represented as fold increase with respect to DMEM ( set as 1 ± SD ) . Data presented were analyzed using two ‐ sided Student ' s t ‐ test : * P < 0 . 05 .

O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O 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-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunoblot (top) and quantification (bottom) of hMENA∆v6 and α-SMA expression in P-CAFs #110 treated with DMEM medium (-) or with conditioned medium (CM) derived from siCNT (siCNT PANC-1) or sihMENA(t) PANC-1 cells ((sihMENA(t) PANC-1) (n = 3). Data are represented as fold increase with respect to DMEM (set as 1± SD). Data presented were analyzed using two‐sided Student's t‐test: *P<0.05.


A . Heat map of proteins identified by LC - MS / MS analysis as differentially secreted in CM of normal fibroblasts ( P - NF # 1 and P - NF # 2 ) , CAFs with low level of hMENA ∆ v6 expression ( P - CAFs low # 44 and # 49 ) , CAFs with high level of hMENA ∆ v6 expression ( P - CAFs high # 67 and # 36 ) . The boxed hMENA ∆ v6 associated signature represents proteins exclusively present in the CM derived from CAFs high . Color code is shown in the upper left corner . Co - variance = 0 . 2 , q value = 0 . 1 ( 10 % false discovery rate ) , adjusted P value = 0 . 0074542 . N = 36 . The values of mass spec identified for each protein were plotted in log2 scale .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Heat map of proteins identified by LC-MS/MS analysis as differentially secreted in CM of normal fibroblasts (P-NF#1 and P-NF#2), CAFs with low level of hMENA∆v6 expression (P-CAFs low#44 and #49), CAFs with high level of hMENA∆v6 expression (P-CAFs high #67 and #36). The boxed hMENA∆v6 associated signature represents proteins exclusively present in the CM derived from CAFs high. Color code is shown in the upper left corner. Co-variance = 0.2, q value = 0.1 (10% false discovery rate), adjusted P value = 0.0074542. N = 36. The values of mass spec identified for each protein were plotted in log2 scale.


B . Quantification of GAS6 secretion levels , as detected by ELISA , in the CM of P - NF ( # 1 ) , P - CAFlow ( # 44 and # 49 ) and P - CAF high ( # 67 and # 36 ) . Data are presented as the mean ± SD of two biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 .

O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of GAS6 secretion levels, as detected by ELISA, in the CM of P-NF (#1), P-CAFlow (#44 and #49) and P-CAF high (#67 and #36). Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.


C . Real time qRT - PCR analysis of the relative GAS6 mRNA expression level in NFs ( P - NF # 1 ) , P - CAF low and P - CAF high , as described above . Data are presented as the mean ± SD of three replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 001 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in NFs (P-NF#1), P-CAF low and P-CAF high, as described above. Data are presented as the mean ± SD of three replicates. Statistical analysis was performed with one-way ANOVA P=0.001, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.


D . Real time qRT - PCR analysis of the relative GAS6 mRNA expression level in PANC - 1 and P - CAF ( n = 3 ) , showing a low GAS6 expression in PANC - 1 cells . Data are presented as the mean ± SD . P values were calculated by two ‐ sided Student ' s t ‐ test . * * * P < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O 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. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in PANC-1 and P-CAF (n=3), showing a low GAS6 expression in PANC-1 cells. Data are presented as the mean ± SD. P values were calculated by two‐sided Student's t‐test. ***P<0.001.


E . Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts ( white ) ( n = 15 ) versus primary NSCLC fibroblasts ( light blue ) ( n = 15 ) ( GSE22862 data set ) . In each boxplot the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) are shown . Statistical significance was calculated by Mann - Whitney U - test ( two - sided ) ( P = 0 . 0208 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O O O O B-DISEASE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts (white) (n=15) versus primary NSCLC fibroblasts (light blue) (n=15) (GSE22862 data set). In each boxplot the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line) are shown. Statistical significance was calculated by Mann-Whitney U-test (two-sided) (P=0.0208).


F , G . Real time qRT - PCR analysis of P # 138 CAF ( F ) and L # 189 CAF ( G ) transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) as representative cases . The siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells ( set as 100 ) . Data are presented as the mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ test * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G. Real time qRT-PCR analysis of P#138 CAF (F) and L#189 CAF (G) transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)) as representative cases. The siRNA‐mediated knock‐down of hMENA/hMENA∆v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells (set as 100). Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test **P<0.01, ***P<0.001.


H . Quantification of matrigel invasion assay of PANC - 1 cultured for 48 h with DMEM ( culture medium ) , or conditioned medium ( CM ) derived from control siRNA P # 106 CAFs ( siCNT P - CAF - CM ) , GAS6 siRNA ( siGAS6 P - CAF - CM ) , hMENA ( t ) siRNA ( sihMENA ( t ) P - CAF - CM ) , hMENA ( t ) siRNA plus rGAS6 ( sihMENA ( t ) P - CAF - CM + rGAS6 ) . Number of invaded PANC - 1 cells after 48 h of treatment was measured by counting 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 004 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of matrigel invasion assay of PANC-1 cultured for 48 h with DMEM (culture medium), or conditioned medium (CM) derived from control siRNA P#106 CAFs (siCNT P-CAF-CM), GAS6 siRNA (siGAS6 P-CAF-CM), hMENA(t) siRNA (sihMENA(t) P-CAF-CM), hMENA(t) siRNA plus rGAS6 (sihMENA(t) P-CAF-CM +rGAS6). Number of invaded PANC-1 cells after 48 h of treatment was measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.004, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.


A . Immunoblot of AXL expression in PANC - 1 and A549 cancer cells upon transfection of control siRNA ( CNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that the knock ‐ down of hMENA isoforms , resulted in a reduction of AXL protein expression .

O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O

A. Immunoblot of AXL expression in PANC-1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression.


B . Real time qRT - PCR analysis of the relative AXL mRNA expression in PANC - 1 and A549 cancer cells transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that the knock ‐ down of hMENA isoforms , resulted in a significant reduction of mRNA AXL expression . Data are presented as the mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ test . * * P < 0 . 01 , * * * P < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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. Real time qRT-PCR analysis of the relative AXL mRNA expression in PANC-1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test. **P<0.01, ***P<0.001.


C . Real time qRT - PCR analysis of the relative levels of mature AXL mRNA or AXL pre - mRNA in PANC - 1 ( left ) and A549 ( right ) cell lines , transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA , sihMENA ( t ) . Data represent percent of AXL mRNA or pre - mRNA levels in hMENA ( t ) silenced cells relative to siCNT control cells ( set at 100 ) . Data are presented as the mean ± SD of two biological replicates . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Real time qRT-PCR analysis of the relative levels of mature AXL mRNA or AXL pre-mRNA in PANC-1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre-mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t‐test. *P< 0.05, ***P<0.001.


D . Immunoblot analysis with the indicated antibodies of PANC - 1 cells transfected with control siRNA ( CNT ) or hMENA ( t ) siRNA , showing that the knock ‐ down of total hMENA isoforms , ( hMENA ( t ) ) inhibits GAS6 - mediated pAXL and pAKT expression . Cells were serum starved overnight and subsequently stimulated with DMSO ( 0 . 02 % ) in control culture medium ( - ) or rGAS6 ( 200 ng / mL ) , for 30 and 60 min . The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported .

O O B-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O

D. Immunoblot analysis with the indicated antibodies of PANC-1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6-mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (-) or rGAS6 (200 ng/mL), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported.


E . Quantification of in vitro matrigel invasion assay of PANC - 1 siCNT cells ( siCNT ) and hMENA / hMENA ∆ v6 silenced cells ( sihMENA ( t ) ) toward rGAS6 as chemo - attractant ( siCNT + rGAS6 and sihMENA ( t ) + rGAS6 ) , showing that the knock ‐ down of hMENA ( t ) reduced cancer cell invasion toward GAS6 . The number of invading cells were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * P < 0 . 01 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)) toward rGAS6 as chemo-attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. **P<0.01.


F . Quantification of in vitro matrigel invasion assay of PANC - 1 siCNT cells ( siCNT ) and hMENA / hMENA ∆ v6 silenced cells ( sihMENA ( t ) ) , untreated ( - ) or treated with conditioned media derived from CAFs ( P - CAF # 36 - CM ) for 48 h . The number of invaded cells were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)), untreated (-) or treated with conditioned media derived from CAFs (P-CAF#36-CM) for 48 h. The number of invaded cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. *** P< 0.001.


A . Overall survival ( OS ) curves in Pancreatic Adenocarcinoma patients ( PDAC ) ( n = 172 ) from The Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 and ENAH ( hMENA ) , is a prognostic signature in PDAC .

O O O B-EXP_ASSAY O O O O O B-DISEASE I-DISEASE O O B-DISEASE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-DISEASE O

A. Overall survival (OS) curves in Pancreatic Adenocarcinoma patients (PDAC) (n=172) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in PDAC.


B . Overall survival ( OS ) curves in Lung Squamous cancer patients ( LUSC ) ( n = 501 ) from The Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 and ENAH ( hMENA ) , is a prognostic signature in LUSC .

O O O B-EXP_ASSAY O O O O O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-DISEASE O

B. Overall survival (OS) curves in Lung Squamous cancer patients (LUSC) (n=501) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in LUSC.


Components of the RLCco - precipitate with ER markers in density gradient fractionation . The post - nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient . ER markers Calnexin , Ribophorin I and Climp63 , co - sedimented with ribosomes ( RPS6 , ribosomal protein subunit 6 ) , mitochondria ( Tim23 ) and RLC proteins at fractions 3 - 5 . The bottom graph shows the density of each fraction .

O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

Components of the RLCco-precipitate with ER markers in density gradient fractionation. The post-nuclear supernatant of cortical neurons was loaded underneath a continuous Optiprep density gradient. ER markers Calnexin, Ribophorin I and Climp63, co-sedimented with ribosomes (RPS6, ribosomal protein subunit 6), mitochondria (Tim23) and RLC proteins at fractions 3-5. The bottom graph shows the density of each fraction.


B ) TRBP , PACT and Ago2 partially co - localize with the ER marker mCherry - CLIMP63 . Hippocampal neurons ( 10 DIV ) expressing mCherry - CLIMP63 were immunostained with TRBP , PACT or Ago2 antibodies . Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters ( scale bars ; 10μm ) .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O

B) TRBP, PACT and Ago2 partially co-localize with the ER marker mCherry-CLIMP63. Hippocampal neurons (10 DIV) expressing mCherry-CLIMP63 were immunostained with TRBP, PACT or Ago2 antibodies. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 10μm).


C ) GFP - Dicer partially co - localizes with mCherry - CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons . Neurons were imaged at 10 DIV using total internal reflection fluorescence ( TIRF ) microscopy using a penetration depth of 150nm . Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters ( scale bars ; 2μm or 10μm ) .

O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O

C) GFP-Dicer partially co-localizes with mCherry-CLIMP63 at the neuronal soma and primary dendrites of hippocampal neurons. Neurons were imaged at 10 DIV using total internal reflection fluorescence (TIRF) microscopy using a penetration depth of 150nm. Boxed insets are magnifications of primary dendrites as depicted by the adjacent letters (scale bars; 2μm or 10μm).


D ) The majority of Dicer , TRBP and PACT are associated with neuronal membranes . Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons . The relative quantity of depicted proteins was analyzed in three independent experiments ( p < 0 . 005 , t - test type 3 , n = 3 , error bars s . d . ) . Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions , respectively .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O

D) The majority of Dicer, TRBP and PACT are associated with neuronal membranes. Sequential detergent extraction was used to separate cytoplasmic from membrane fractions in 7 DIV cortical neurons. The relative quantity of depicted proteins was analyzed in three independent experiments (p<0.005, t-test type 3, n=3, error bars s.d.). Actin and ribophorin I were used as loading controls for cytoplasmic and membrane fractions, respectively.


E ) TRBP is cytoplasmic in primary astrocytes . Sequential detergent extraction was performed in primary astrocyte cultures . Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions , respectively .

O O B-GENEPROD O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O

E) TRBP is cytoplasmic in primary astrocytes. Sequential detergent extraction was performed in primary astrocyte cultures. Actin and calnexin were used as loading controls for cytoplasmic and membrane fractions, respectively.


F ) The majority of Dicer , TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells , isolated using sequential detergent extraction .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O B-CELL_LINE O O O O O O O O

F) The majority of Dicer, TRBP and PACT proteins are present in cytoplasmic fractions in HEK293T cells, isolated using sequential detergent extraction.


A ) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes . Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation . Protein intensity values were normalized to calnexin in each of three independent experiments ( p = 0 . 008 , t - test type 3 , n = 3 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O 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) BDNF stimulation leads to a decrease in TRBP levels from ER microsomes. Cortical neurons were stimulated BDNF with or vehicle control and ER microsomes were isolated using differential centrifugation. Protein intensity values were normalized to calnexin in each of three independent experiments (p=0.008, t-test type 3, n=3, error bars; s.d.).


B ) Endogenous TRBP partially co - localizes with calnexin at the neuronal cell soma and primary dendrites . Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation . Boxed insets are magnifications of the primary dendrite ( 15μm in length ) . Arrows point to overlapping puncta ( scale bars ; 20μm ( GFP ) or 10μm ) .

O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O

B) Endogenous TRBP partially co-localizes with calnexin at the neuronal cell soma and primary dendrites. Hippocampal neurons were transfected with GFP at 7DIV and fixed at 10DIV following BDNF or control stimulation. Boxed insets are magnifications of the primary dendrite (15μm in length). Arrows point to overlapping puncta (scale bars; 20μm (GFP) or 10μm).


C ) BDNF leads to a decrease in co - localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons . Manders co - localization co - efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites ( 15μm ) using GFP as a morphological mask . Average co - localization values were normalized to controls in each experiment ( p < 0 . 008 , t - test type 3 , n = 4 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O 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) BDNF leads to a decrease in co-localization between TRBP and calnexin at the soma and primary dendrites of hippocampal neurons. Manders co-localization co-efficients between TRBP and Calnexin were measured in the cell soma or primary dendrites (15μm) using GFP as a morphological mask. Average co-localization values were normalized to controls in each experiment (p<0.008, t-test type 3, n=4, error bars; s.d.).


D ) BDNF decreases mCherry - CLIMP63 co - localization with TRBP , but not with PACT or Ago2 . Hippocampal neurons were transfected with GFP and mCherry - CLIMP63 at 7DIV and immunostained three days later , following BDNF or control stimulation . Manders co - localization was measured at the cell soma using GFP as a mask as indicated ( p = 0 . 01 , t - test type 3 , n = 4 , error bars ; s . d . ) .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O 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) BDNF decreases mCherry-CLIMP63 co-localization with TRBP, but not with PACT or Ago2. Hippocampal neurons were transfected with GFP and mCherry-CLIMP63 at 7DIV and immunostained three days later, following BDNF or control stimulation. Manders co-localization was measured at the cell soma using GFP as a mask as indicated (p=0.01, t-test type 3, n=4, error bars; s.d.).


E ) BDNF leads to an increase in the levels of TRBP ( p = 0 . 03 ) and PACT ( p = 0 . 004 ) in the cytoplasmic fraction . Cortical neurons ( 7 DIV ) were control - or BDNF - stimulated and cytoplasmic fractions were isolated using digitonin . Protein intensity values were normalized to actin in each experiment . The position of the star corresponds to the TRBP band used for analysis ( ~ 43kDa ) ( t - test type 3 , n = 4 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O 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) BDNF leads to an increase in the levels of TRBP (p=0.03) and PACT (p=0.004) in the cytoplasmic fraction. Cortical neurons (7 DIV) were control- or BDNF-stimulated and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each experiment. The position of the star corresponds to the TRBP band used for analysis (~43kDa) (t-test type 3, n=4, error bars; s.d.).


F ) The BDNF - induced increase in cytoplasmic TRBP is dependent on intracellular calcium . Cortical neurons ( 7 DIV ) were treated with vehicle control , BDNF and BAPTA - AM as indicated , and cytoplasmic fractions were isolated using digitonin . Protein intensity values were normalized to actin in each independent experiment ( p = 0 . 03 , n = 3 , error bars ; s . d . ) .

O O O B-GENEPROD O O O O B-SUBCELLULAR B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD 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) The BDNF-induced increase in cytoplasmic TRBP is dependent on intracellular calcium. Cortical neurons (7 DIV) were treated with vehicle control, BDNF and BAPTA-AM as indicated, and cytoplasmic fractions were isolated using digitonin. Protein intensity values were normalized to actin in each independent experiment (p=0.03, n=3, error bars; s.d.).


BDNF stimulation causes a decrease in the TRBP - Dicer interaction . Young cortical neurons were treated with BDNF or vehicle control and co - immunoprecipitations ( co - IPs ) were performed using 2μg control IgG or polyclonal anti - TRBP antibodies . Dicer binding in co - IP was normalized to inputs in each experiment ( p = 0 . 001 , t - test type 2 , n = 3 , error bars represent s . d . ) .

B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BDNF stimulation causes a decrease in the TRBP-Dicer interaction. Young cortical neurons were treated with BDNF or vehicle control and co-immunoprecipitations (co-IPs) were performed using 2μg control IgG or polyclonal anti-TRBP antibodies. Dicer binding in co-IP was normalized to inputs in each experiment (p=0.001, t-test type 2, n=3, error bars represent s.d.).


B ) Short BDNF stimulation changes the levels of only a few microRNAs , such as miR - 16 - 5p and miR - 551b - 5p . Volcano plot of small RNA sequencing data from BDNF - or control - treated cortical neurons ( 6DIV ) was based on differential expression analysis using the edgeR package . Individual points represent the average fold change obtained in three independent experiments for each miRNA , and plotted against obtained p - values . MiRNAs above the red dashed line are significant ( p < 0 . 05 , t - test type 2 , n = 3 ) .

O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Short BDNF stimulation changes the levels of only a few microRNAs, such as miR-16-5p and miR-551b-5p. Volcano plot of small RNA sequencing data from BDNF- or control-treated cortical neurons (6DIV) was based on differential expression analysis using the edgeR package. Individual points represent the average fold change obtained in three independent experiments for each miRNA, and plotted against obtained p-values. MiRNAs above the red dashed line are significant (p<0.05, t-test type 2, n=3).


C ) BDNF stimulation leads to a transient decrease in mature miR - 16 - 5p levels . 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR . Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation ( n = 3 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O 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) BDNF stimulation leads to a transient decrease in mature miR-16-5p levels. 6DIV cortical neurons were treated with BDNF for 10 or 20 minutes and mature miRNA levels were measured in rtPCR. Ct values for each miRNA were normalized to U6 and to 20 minutes of control stimulation (n=3, p=0.02, t-test type 3, error bars; s.d.).


D ) BDNF stimulation leads to a transient increase in the levels of pre - miR16 after 10 minutes of BDNF stimulation . Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation ( n = 4 , t - test type 3 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O 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) BDNF stimulation leads to a transient increase in the levels of pre-miR16 after 10 minutes of BDNF stimulation. Ct values obtained in rtPCR were normalized to U6 and to 20 minutes control stimulation (n=4, t-test type 3, error bars; s.d.).


E ) BDNF causes a decrease in pre - miR16 binding to TRBP . Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti - TRBP antibody . Ct values measured in rtPCR were normalized to respective inputs ( n = 3 , p = 0 . 002 , t - test type 3 , error bars represent s . d . ) .

O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O 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) BDNF causes a decrease in pre-miR16 binding to TRBP. Cortical neurons were treated with BDNF or vehicle control and RNA IPs were performed using a mouse monoclonal anti-TRBP antibody. Ct values measured in rtPCR were normalized to respective inputs (n=3, p=0.002, t-test type 3, error bars represent s.d.).


A ) BDNF changes the canonical isomiR proportion of a subset of miRNAs . IsomiR levels were compared to the total amount of the corresponding miRNA . Shown are the miRNAs that had significant changes in the canonical isomiR ( annotated as ' 0 ' , p < 0 . 05 , t - test type 2 , n = 3 ) . Results are shown in order of decreasing significance . Shown in red are miRNAs of the miR16 family , miR - 16 - 5p and miR - 15b - 5p . Black squares correspond non - existing isomiRs . Numbers on the top of the table represent the trimming ( negative ) or addition ( positive ) of nucleotides to the 3 ' - or 5 ' - end of the canonical 5p or 3p isomiRs , respectively . Only templated nucleotide additions and trimmings , which might reflect Dicer - mediated pre - miRNA cleavage , were considered for analysis .

O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

A) BDNF changes the canonical isomiR proportion of a subset of miRNAs. IsomiR levels were compared to the total amount of the corresponding miRNA. Shown are the miRNAs that had significant changes in the canonical isomiR (annotated as '0', p<0.05, t-test type 2, n=3). Results are shown in order of decreasing significance. Shown in red are miRNAs of the miR16 family, miR-16-5p and miR-15b-5p. Black squares correspond non-existing isomiRs. Numbers on the top of the table represent the trimming (negative) or addition (positive) of nucleotides to the 3'- or 5'-end of the canonical 5p or 3p isomiRs, respectively. Only templated nucleotide additions and trimmings, which might reflect Dicer-mediated pre-miRNA cleavage, were considered for analysis.


B ) BDNF stimulation leads to a decrease in the canonical ( 22 nucleotides ) isomiRs of the miR16 family , miR - 16 - 5p ( p = 0 . 005 ) and miR - 15b - 5p ( p = 0 . 02 ) . In both cases , this is accompanied by an increase in the levels of shorter isomiRs ; the 19 - nucleotide isoform of miR - 16 - 5p ( p = 0 . 04 ) and the 21 - nucleotide isoform of miR - 15b - 5p ( p = 0 . 007 ) . The relative isomiRs proportion was calculated as described in A ( t - test type 2 , n = 3 , error bars ; s . d . ) .

O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O 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) BDNF stimulation leads to a decrease in the canonical (22 nucleotides) isomiRs of the miR16 family, miR-16-5p (p=0.005) and miR-15b-5p (p=0.02). In both cases, this is accompanied by an increase in the levels of shorter isomiRs; the 19-nucleotide isoform of miR-16-5p (p=0.04) and the 21-nucleotide isoform of miR-15b-5p (p=0.007). The relative isomiRs proportion was calculated as described in A (t-test type 2, n=3, error bars; s.d.).


B ) Endogenous miR - 16 - 5p activity in developing hippocampal neurons . Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ( ' LNA Ctrl ' ) or the anti - sense miR - 16 - 5p inhibitor ( ' LNA - miR16 ' ) . Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ( ' GFP - only ' ) was compared to the total number of transfected neurons . LNA - miR16 reduces the amount of repression of the miR16 sensor compared to LNA - Ctrl ( n = 4 , p = 0 . 02 , t - test type 2 , error bars represent s . d . ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O 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) Endogenous miR-16-5p activity in developing hippocampal neurons. Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ('LNA Ctrl') or the anti-sense miR-16-5p inhibitor ('LNA-miR16'). Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ('GFP-only') was compared to the total number of transfected neurons. LNA-miR16 reduces the amount of repression of the miR16 sensor compared to LNA-Ctrl (n=4, p=0.02, t-test type 2, error bars represent s.d.).


C ) Short - term BDNF stimulation inhibits endogenous miR - 16 - 5p activity . Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B . Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment ( n = 4 , p = 0 . 02 , t - test type 3 , error bars ; s . d . ) .

O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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) Short-term BDNF stimulation inhibits endogenous miR-16-5p activity. Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B. Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.).