D . Measurements by RT - PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild - type mice ( RBEWT / WT ) , KI - mice expressing the RIBEYE - A - domain fusion protein with GCamP3 ( RBEKI / KI ) , and KO mice ( RBEKO / KO ) . These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina , and to examine whether the RIBEYE A - domain KO impairs CtBP2 expression in brain , which expresses little RIBEYE . Assays are based on a qRT - PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms ( RIBEYE and CtBP2 ) . Statistical analyses were performed using Student ' s t - test ; data are presented as mean ± SEM ( * * * , p < 0 . 001 ; n = 4 mice per genotype ) .

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

D. Measurements by RT-PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild-type mice (RBEWT/WT), KI-mice expressing the RIBEYE-A-domain fusion protein with GCamP3 (RBEKI/KI), and KO mice (RBEKO/KO). These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina, and to examine whether the RIBEYE A-domain KO impairs CtBP2 expression in brain, which expresses little RIBEYE. Assays are based on a qRT-PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms (RIBEYE and CtBP2). Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (***, p<0.001; n=4 mice per genotype).


E . Representative immunofluorescence labeling of cryostat sections from littermate wild - type and RBEKI / KI mice for EGFP ( green , to detect GCamP3 in RIB - G3 ) and SV2 ( red , to label synapses ) demonstrates expression of the A - domain / GCamP3 fusion protein in knockin mice ( OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , granule cell layer ) . Scale bar = 5 µm .

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E. Representative immunofluorescence labeling of cryostat sections from littermate wild-type and RBEKI/KI mice for EGFP (green, to detect GCamP3 in RIB-G3) and SV2 (red, to label synapses) demonstrates expression of the A-domain/GCamP3 fusion protein in knockin mice (OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, granule cell layer). Scale bar = 5 µm.


F . Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT / WT and RBEKO / KO mice . Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies ; results are normalized to loading controls depending on molecular weights ( actin , fodrin , VCP , and GDI ) and to wild - type controls . Statistical analyses were performed using Student ' s t - test ; data are presented as mean ± SEM ( * , p < 0 . 05 ; n = 4 mice per genotype ) .

O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT/WT and RBEKO/KO mice. Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies; results are normalized to loading controls depending on molecular weights (actin, fodrin, VCP, and GDI) and to wild-type controls. Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (*, p<0.05; n=4 mice per genotype).


G . Immunofluoresence labeling for CtBP2 ( green , to label RIBEYE ) and mGluR6 ( red , to label photoreceptorsynapses ) in cryostat sections from littermate RBEWT / WT and RBEKO / KOmouseretinas demonstrates that the KO abolishes RIBEYE expression ( OPL , outer plexiform layer ) . Scale bars : 5 µm .

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

G. Immunofluoresence labeling for CtBP2 (green, to label RIBEYE) and mGluR6 (red, to label photoreceptorsynapses) in cryostat sections from littermate RBEWT/WT and RBEKO/KOmouseretinas demonstrates that the KO abolishes RIBEYE expression (OPL, outer plexiform layer). Scale bars: 5 µm.


A . Overview of the synaptic organization of the retina in RBEWT / WT and RBEKO / KOmice . Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B - domain ( CtBP2 ; green ) and SV2 . Scale bar : 20 µm ; abbreviations : ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , ganglion cell layer .

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

A. Overview of the synaptic organization of the retina in RBEWT/WT and RBEKO/KOmice. Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B-domain (CtBP2; green) and SV2. Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


B . Double immunofluorescence staining of retinas from RBEWT / WT and RBEKO / KO mice for PSD95 ( green , to label postsynaptic specializations ) and mGluR6 ( red , to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses ) . Abbreviations are as in A . Scale bars : 20 µm ( left and right overview ) , 5 µm ( left panel magnification ) , 2 µm ( right panel magnification ) . C . Same as B , except that retinas were stained for PKCα ( green , to label bipolar neurons ) and PSD95 ( red , to label amacrine cells ) .

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

B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice for PSD95 (green, to label postsynaptic specializations) and mGluR6 (red, to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses). Abbreviations are as in A. Scale bars: 20 µm (left and right overview), 5 µm (left panel magnification), 2 µm (right panel magnification).C. Same as B, except that retinas were stained for PKCα (green, to label bipolar neurons) and PSD95 (red, to label amacrine cells).


A and B . Double immunofluorescence staining of retinas from RBEWT / WT and RBEKO / KO mice , respectively , for PSD95 ( green , to label presynaptic photoreceptor terminals ) and Cav1 . 4 ( red , to label presynaptic Ca2 + - channel ) . Scale bar : 20 µm ; abbreviations : ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer . ( A1 to A4 ) Magnification of Cav1 . 4 staining in the outer plexiform layer ( OPL ) in the retina of RBEWT / WT and ( B1 to B4 ) RBEKO / KO mice . White arrow heads indicate Ca2 + - channel distribution at selected synapses . Images represent 2 µm z - stack projections . Scale bar : 2 µm .

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A and B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice, respectively, for PSD95 (green, to label presynaptic photoreceptor terminals) and Cav1.4 (red, to label presynaptic Ca2+-channel). Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (A1 to A4) Magnification of Cav1.4 staining in the outer plexiform layer (OPL) in the retina of RBEWT/WT and (B1 to B4) RBEKO/KO mice. White arrow heads indicate Ca2+-channel distribution at selected synapses. Images represent 2 µm z-stack projections. Scale bar: 2 µm.


A . Transmission EM demonstrates the absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in the terminals of RBEWT / WTmice . Asterisks in A2 and A3 denote photoreceptor active zones of RBEKO / KOmice in which the synaptic ribbon is absent . Except for the absence of synaptic ribbons , the ultrastructural appearance of the presynaptic terminals is comparable to that of RBEWT / WTmice . Abbreviations : sr , synaptic ribbon ; pre , presynaptic ; post , postsynaptic . Scale bars : 500 nm .

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

A. Transmission EM demonstrates the absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Asterisks in A2 and A3 denote photoreceptor active zones of RBEKO/KOmice in which the synaptic ribbon is absent. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to that of RBEWT/WTmice. Abbreviations: sr, synaptic ribbon; pre, presynaptic; post, postsynaptic. Scale bars: 500 nm.


B . Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in the terminals of RBEWT / WTmice . Except for the absence of synaptic ribbons , the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT / WTmice . Abbreviations are the same as in A . Scale bars : 1 µm ( B1 , B2 , B4 , B5 ) ; 300 nm ( B3 , B6 ) .

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

B. Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT/WTmice. Abbreviations are the same as in A. Scale bars: 1 µm (B1, B2, B4, B5); 300 nm (B3, B6).


A and B . Representative transmission EM pictures of RBEWT / WT ( A ) and RBEKO / KO ( B ) photoreceptor ribbon synapses ( blue rectangles , area adjacent to synaptic junction used for measuring junction - associated vesicle density [ size = 200 nm x 300 nm ] ; black squares , are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [ size = 200 nm x 200 nm ] ) . C . Average vesicle ratio ( expressed as vesicle numbers in the area adjacent to synaptic junctions ( blue rectangle ) or the cytosol ( black square ) normalized to RBEWT / WT . D . Average number of docked vesicles within a 150 nm range in the vicinity of the active zone .

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

A and B. Representative transmission EM pictures of RBEWT/WT (A) and RBEKO/KO (B) photoreceptor ribbon synapses (blue rectangles, area adjacent to synaptic junction used for measuring junction-associated vesicle density [size = 200 nm x 300 nm]; black squares, are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [size = 200 nm x 200 nm]).C. Average vesicle ratio (expressed as vesicle numbers in the area adjacent to synaptic junctions (blue rectangle) or the cytosol (black square) normalized to RBEWT/WT.D. Average number of docked vesicles within a 150 nm range in the vicinity of the active zone.


A . Experimental design and representative traces for analysis of synaptic transmission in retinas from wild - type ( black ) and RIBEYE KO mice ( red ) . Presynaptic bipolar neurons were depolarized in voltage - clamp mode from - 70 mV to - 10 mV for 0 . 5 s as indicated on top to open Ca2 + - channels and trigger release . Presynaptic Ca2 + - currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces ; dotted box displays an expansion of the initial Ca2 + - currents and EPSCs . B . RIBEYE KO does not alter presynaptic Ca2 + - currents . Summary graphs show average peak Ca2 + - currents and Ca2 + - current . C . RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization . Plot shows integrated EPSC charge as a function of time ; both initial and sustained neurotransmitter release are impaired . D . RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude . E . RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization . Summary graphs display integrated EPSC charges for the initial phase ( 0 - 50 ms ) and the sustained phase ( 50 - 500 ms ) . F . RIBEYE KO does not alter the kinetics of EPSCs . Summary graphs depict the mean synaptic delays ( left ) , rise times ( center ) and decay time constants ( right ) of EPSCs .

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

A. Experimental design and representative traces for analysis of synaptic transmission in retinas from wild-type (black) and RIBEYE KO mice (red). Presynaptic bipolar neurons were depolarized in voltage-clamp mode from -70 mV to -10 mV for 0.5 s as indicated on top to open Ca2+-channels and trigger release. Presynaptic Ca2+-currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces; dotted box displays an expansion of the initial Ca2+-currents and EPSCs.B. RIBEYE KO does not alter presynaptic Ca2+-currents. Summary graphs show average peak Ca2+-currents and Ca2+-current.C. RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization. Plot shows integrated EPSC charge as a function of time; both initial and sustained neurotransmitter release are impaired.D. RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude.E. RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization. Summary graphs display integrated EPSC charges for the initial phase (0-50 ms) and the sustained phase (50-500 ms).F. RIBEYE KO does not alter the kinetics of EPSCs. Summary graphs depict the mean synaptic delays (left), rise times (center) and decay time constants (right) of EPSCs.


A . Representative mEPSCs recorded from AII cells in wild - type ( black ) and RIBEYE KO mice ( red ) . The insets in dashed boxes show mEPSCs on expanded scales , while the inset in a continuous box on the right shows the averaged mEPSC waveforms ( average of 100 - 200 isolated mini events ) of the example cells . Calibration bars apply to wild - type and mutant EPSCs . B . RIBEYE KO does not alter fundamental parameters of mEPSCs . Summary graphs show mean mEPSC frequency , amplitude , integrated charge , rise time , and decay time constant . Note that the only excitatory inputs into AII amacrine cells is provided by bipolar cell synapses .

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

A. Representative mEPSCs recorded from AII cells in wild-type (black) and RIBEYE KO mice (red). The insets in dashed boxes show mEPSCs on expanded scales, while the inset in a continuous box on the right shows the averaged mEPSC waveforms (average of 100-200 isolated mini events) of the example cells. Calibration bars apply to wild-type and mutant EPSCs.B. RIBEYE KO does not alter fundamental parameters of mEPSCs. Summary graphs show mean mEPSC frequency, amplitude, integrated charge, rise time, and decay time constant. Note that the only excitatory inputs into AII amacrine cells is provided by bipolar cell synapses.


C and D . RIBEYE KO renders mEPSCs sensitive to the slow Ca2 + - buffer EGTA ( C , representative mEPSCs traces recorded from AII cells after 30 minutes incubation in DMSO or EGTA - AM [ 0 . 2 mM ] ; D , summary graphs of the mean mEPSC frequency ( left ) and amplitude ( right ) after incubation in DMSO or EGTA - AM ) .

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C and D. RIBEYE KO renders mEPSCs sensitive to the slow Ca2+-buffer EGTA (C, representative mEPSCs traces recorded from AII cells after 30 minutes incubation in DMSO or EGTA-AM [0.2 mM]; D, summary graphs of the mean mEPSC frequency (left) and amplitude (right) after incubation in DMSO or EGTA-AM).


E and F . mEPSCs in AII amacrine cells are largely triggered by Ca2 + - influx via presynaptic L - type Ca2 + - channels ( E , representative mEPSCs traces recorded in regular [ 2 mM ] or reduced extracellular Ca2 + - concentration [ 0 . 3 mM ] , or in the presence of nimodipine [ 50 µM ] in regular Ca2 + ; F , summary graphs of the mean mEPSC frequency and amplitude under the conditions described for E ) .

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E and F. mEPSCs in AII amacrine cells are largely triggered by Ca2+-influx via presynaptic L-type Ca2+-channels (E, representative mEPSCs traces recorded in regular [2 mM] or reduced extracellular Ca2+-concentration [0.3 mM], or in the presence of nimodipine [50 µM] in regular Ca2+; F, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for E).


G and H . mEPSCs in AII amacrine cells are suppressed by the fast Ca2 + - buffer BAPTA ( G , representative mEPSCs traces recorded after 30 min incubations with DMSO or BAPTA - AM [ 30 µM ] ; H , summary graphs of the mean mEPSC frequency and amplitude under the conditions described for G ) .

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G and H. mEPSCs in AII amacrine cells are suppressed by the fast Ca2+-buffer BAPTA (G, representative mEPSCs traces recorded after 30 min incubations with DMSO or BAPTA-AM [30 µM]; H, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for G).


Autophagy components promote CHIKV infection and control virus ‐ induced cell death in HeLa cells . Cells were mock infected or infected and immunoblotted for actin , p62 ( A ) or LC3 ( B ) .

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Autophagy components promote CHIKV infection and control virus‐induced cell death in HeLa cells. Cells were mock infected or infected and immunoblotted for actin, p62 (A) or LC3 (B).


Cells were infected for 15 h and labeled using antibodies to p62 and capsid ( C ) .

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Cells were infected for 15 h and labeled using antibodies to p62 and capsid (C).


Cells were transfected with GFP ‐ LC3 ‐ B , infected for 15 h and labeled with anti ‐ capsid antibody ( D ) .

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Cells were transfected with GFP‐LC3‐B, infected for 15 h and labeled with anti‐capsid antibody (D).


Mice were infected for 3 days , then whole ‐ cell lysates of muscle were immunoblotted for LC3 or actin ( E ) and muscle sections were labeled with p62 and capsid antibodies ( F ) .

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Mice were infected for 3 days, then whole‐cell lysates of muscle were immunoblotted for LC3 or actin (E) and muscle sections were labeled with p62 and capsid antibodies (F).


Cells were treated with DMSO ( CTRL ) or rapamycin and then infected . Viral replication ( G ) and production ( H ) were assessed and whole ‐ cell lysates were immunoblotted for capsid or actin ( I ) .

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Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I).


Cells were treated with CTRL , Beclin1 ( left ) or Atg7 ( right ) siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) , viral replication ( K ) and production ( L ) were assessed and whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for Beclin1 , Atg7 , capsid and 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 . CHIKV , Chikungunya virus ; CTRL , control ; GFP , green fluorescent protein ; DMSO , dimethylsulphoxide ; siRNA , short interfering RNA .

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Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells were immunoblotted for Beclin1, Atg7, capsid and 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. CHIKV, Chikungunya virus; CTRL, control; GFP, green fluorescent protein; DMSO, dimethylsulphoxide; siRNA, short interfering RNA.


Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells . Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid ( A ) , to p62 , NDP52 and capsid ( B ) , to NDP52 , LC3 ‐ C or capsid ( C ) , to p62 , ubiquitin ( FK2 ) and capsid ( D ) , or to NDP52 , ubiquitin ( FK2 ) , and capsid ( E ) .

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Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells. Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid (A), to p62, NDP52 and capsid (B), to NDP52, LC3‐C or capsid (C), to p62, ubiquitin (FK2) and capsid (D), or to NDP52, ubiquitin (FK2), and capsid (E).


Cells were treated with CTRL or p62 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( F ) , viral replication ( left ) and qRT ‐ PCR ( right ) ( G ) , and viral production ( H ) were assessed and whole ‐ cell lysates were immunoblotted for p62 , capsid or actin ( I ) .

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Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (F), viral replication (left) and qRT‐PCR (right) (G), and viral production (H) were assessed and whole‐cell lysates were immunoblotted for p62, capsid or actin (I).


Cells were treated with CTRL or NDP52 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) , viral replication ( left ) and qRT ‐ PCR ( right ) ( K ) and production were assessed ( L ) .

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

Cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (left) and qRT‐PCR (right) (K) and production were assessed (L).


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 .

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

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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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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 ) .

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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 ) .

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

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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

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 ) .

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

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 ) .

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

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 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O 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 ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O 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 ) .

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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 .

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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 ) .

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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 ) .

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

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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

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

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

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

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