( f ) Actin accumulates within wrinkles at the E - YSL . The yolk cell membrane of LifeAct - GFP injected embryos ( actin - green ) was stained with lectin - TRITC ( red ) ( top ) . Orthogonal projection of the wrinkled area ( bottom ) . Wrinkles are outlined with a white dotted line . Scale bar 2 μm .

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(f) Actin accumulates within wrinkles at the E-YSL. The yolk cell membrane of LifeAct-GFP injected embryos (actin - green) was stained with lectin-TRITC (red) (top). Orthogonal projection of the wrinkled area (bottom). Wrinkles are outlined with a white dotted line. Scale bar 2 μm.


( a ) Yolk granules flow in stereotyped patterns as epiboly progresses . PIV of time - lapse snapshots imaged by two - photon microscopy of a Tg ( β - actin : m - GFP ) embryo ( from Movie EV2 ) . Upon epiboly onset , yolk granules flow towards the animal pole displaying two internal vortices near the E - YSL . At 50 % , the movement of the yolk granules halts , but resumes afterwards until the end of epiboly . Animal ( A ) and vegetal ( V ) poles are indicated . Scale bar 100 μm . ( b ) Internal yolk content speed ( μm / min ) at the center of the embryo , over time ( min ) . Inset : Spherical coordinates . The main axis of the sphere is represented by z , from the animal to the vegetal pole . The polar angle is represented by φ and the radial coordinate is denoted ρ . ( c ) Vortex midpoints distance from the center ( ρ ) , over time ( min ) . ( d ) Vortex midpoints position in relation to the φ axis , over time ( min ) . Black lines represent individual measurements . Red lines represent averages . ( e ) Correlation between vortex displacement ( blue , average of n = 3 embryos ) vs . EVL margin displacement ( red , average of n = 4 embryos ) along the main axis ( z ) , over time ( min ) . Standard Errors are displayed . Arrow points to 50 % epiboly .

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(a) Yolk granules flow in stereotyped patterns as epiboly progresses. PIV of time-lapse snapshots imaged by two-photon microscopy of a Tg (β-actin:m-GFP) embryo (from Movie EV2). Upon epiboly onset, yolk granules flow towards the animal pole displaying two internal vortices near the E-YSL. At 50 %, the movement of the yolk granules halts, but resumes afterwards until the end of epiboly. Animal (A) and vegetal (V) poles are indicated. Scale bar 100 μm. (b) Internal yolk content speed (μm/min) at the center of the embryo, over time (min). Inset: Spherical coordinates. The main axis of the sphere is represented by z, from the animal to the vegetal pole. The polar angle is represented by φ and the radial coordinate is denoted ρ. (c) Vortex midpoints distance from the center (ρ), over time (min). (d) Vortex midpoints position in relation to the φ axis, over time (min). Black lines represent individual measurements. Red lines represent averages. (e) Correlation between vortex displacement (blue, average of n=3 embryos) vs. EVL margin displacement (red, average of n = 4 embryos) along the main axis (z), over time (min). Standard Errors are displayed. Arrow points to 50 % epiboly.


( a ) Yolk microrheology . Mean squared displacement ( MSD ) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos . Solid and dashed black lines are Mean ± Standard Error computed from 99 particles . Red line is the fit of the Stokes - Einstein equation . MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag , Δt , consistent with a Newtonian liquid behavior with a viscosity of 129 mPa · s . A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila ( He et al . , 2014 ) . Interestingly , the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C . elegansembryos ( Daniels et al . , 2006 ) and of the cytoplasm of several cell types ( Wirtz , 2009 ) .

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(a) Yolk microrheology. Mean squared displacement (MSD) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos. Solid and dashed black lines are Mean ± Standard Error computed from 99 particles. Red line is the fit of the Stokes-Einstein equation. MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag, Δt, consistent with a Newtonian liquid behavior with a viscosity of 129 mPa·s. A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila (He et al., 2014). Interestingly, the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C. elegansembryos (Daniels et al., 2006) and of the cytoplasm of several cell types (Wirtz, 2009).


( b ) Viscoelasticity of the yolk cortex . Effective complex modulus ( g * ) measured by indenting the surface of zebrafishembryos with AFM . Red symbols are the elastic modulus ( g ' ) and yellow symbols are the viscous modulus ( g ' ' ) , respectively . Standard Errors are displayed . In all the measured frequencies cortex rheology is dominated by a solid - like behavior with the viscous modulus 5 times lower that the elastic modulus . No significant frequency dependence was found in the elastic ( ANOVA , p = 0 . 123 ) and viscous modulus ( ANOVA , p = 0 . 719 ) .

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(b) Viscoelasticity of the yolk cortex. Effective complex modulus (g*) measured by indenting the surface of zebrafishembryos with AFM. Red symbols are the elastic modulus (g') and yellow symbols are the viscous modulus (g''), respectively. Standard Errors are displayed. In all the measured frequencies cortex rheology is dominated by a solid-like behavior with the viscous modulus 5 times lower that the elastic modulus. No significant frequency dependence was found in the elastic (ANOVA, p = 0.123) and viscous modulus (ANOVA, p = 0.719).


( c ) Flow lines from snapshots of the PIV measurements ( at 40 , 60 and 80 % epiboly ) of a two - photon video time - lapse ( from Movie EV4 ) of a Tg ( β - actin : m - GFP ) embryo ( top ) . Fitted flow lines using HR ar shown at the bottom . The relative mean square errors ( RMSE ) of the fitting are displayed as a percentage at the bottom left of each panel . Scale bar 100 μm .

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(c) Flow lines from snapshots of the PIV measurements (at 40, 60 and 80 % epiboly) of a two-photon video time-lapse (from Movie EV4) of a Tg (β-actin:m-GFP) embryo (top). Fitted flow lines using HR ar shown at the bottom. The relative mean square errors (RMSE) of the fitting are displayed as a percentage at the bottom left of each panel. Scale bar 100 μm.


( d ) Mechanical power maps over time obtained by HR of the same embryos as in ( c ) ( from Movie EV5 ) . The RMSE of the power is shown as a percentage at the bottom left of each panel . The mechanical work to produce doming ( 40 % epiboly ) arises mainly at the blastoderm ( left ) ; at 60 % epiboly ( middle ) DCs undergo ingression ( red ) while the E - YSL resists constriction ( blue ) . At 80 % epiboly ( right ) , the active work leading to the active movement of the EVL / yolk margin mainly maps at the E - YSL ( red ) while the blastoderm undergoes elastic resistance ( blue ) . White arrows point to the main observed events . Scale bar 100 μm .

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(d) Mechanical power maps over time obtained by HR of the same embryos as in (c) (from Movie EV5). The RMSE of the power is shown as a percentage at the bottom left of each panel. The mechanical work to produce doming (40 % epiboly) arises mainly at the blastoderm (left); at 60 % epiboly (middle) DCs undergo ingression (red) while the E-YSL resists constriction (blue). At 80 % epiboly (right), the active work leading to the active movement of the EVL / yolk margin mainly maps at the E-YSL (red) while the blastoderm undergoes elastic resistance (blue). White arrows point to the main observed events. Scale bar 100 μm.


( e ) AV ( red ) and CC ( green ) stresses and their differences ( blue ) normalized values in arbitrary units from the regression analysis ( Movie EV6 ) were plotted as a function of the φ angle ( 0 - 1 ) along the cortex from animal to vegetal . The equator - dotted yellow line - , yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed ( 40 % ; 60 % ; 80 % epiboly ) . The CC stress steeps up from animal to vegetal and the AV shows a more complex profile . Their difference changes senses around the EVL / yolk margin .

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(e) AV (red) and CC (green) stresses and their differences (blue) normalized values in arbitrary units from the regression analysis (Movie EV6) were plotted as a function of the φ angle (0-1) along the cortex from animal to vegetal. The equator - dotted yellow line -, yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed (40 %; 60 %; 80 % epiboly). The CC stress steeps up from animal to vegetal and the AV shows a more complex profile. Their difference changes senses around the EVL / yolk margin.


( a ) Correlation between the differential tension ( cyan ) represented by the recoil velocities differences ( m / s ) between CC and AV laser cuts and their difference ( blue ) calculated by regression analysis at the E - YSL during epiboly ( % ) . The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points .

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(a) Correlation between the differential tension (cyan) represented by the recoil velocities differences (m/s) between CC and AV laser cuts and their difference (blue) calculated by regression analysis at the E-YSL during epiboly (%). The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points.


( b ) AV tension ( averaged recoil velocity in m / s ) in the yolk cortex at different distances from the EVL ( 0 , 10 , 20 , 40 and 60 m ) and at different epiboly stages ( 50 % , blue ; 60 - 70 % , red ; 80 % epiboly , green ) . Significance changes are highlighted and Standard Errors are displayed .

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(b) AV tension (averaged recoil velocity in m/s) in the yolk cortex at different distances from the EVL (0, 10, 20, 40 and 60 m) and at different epiboly stages (50 %, blue; 60-70 %, red; 80 % epiboly, green). Significance changes are highlighted and Standard Errors are displayed.


( c ) Probing mechanical parameters of living embryos with AFM . Force - indentation curves recorded at cantilever velocity of 10 m / s ( blue ) fitted with a " liquid balloon " model ( red ) . Inset : AFM force - indentation description .

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(c) Probing mechanical parameters of living embryos with AFM. Force-indentation curves recorded at cantilever velocity of 10 m/s (blue) fitted with a "liquid balloon" model (red). Inset: AFM force-indentation description.


( d ) Averaged values of mean surface tension ( pN / μm ) on the yolk obtained with AFM at different epiboly stages ( left ) , and the significant difference of tension at 60 - 70 % epiboly ( p = 0 . 0245 ) between the E - YSL and the vegetal pole ( right ) . Standard Errors are displayed .

(d) Averaged values of mean surface tension (pN / μm) on the yolk obtained with AFM at different epiboly stages (left), and the significant difference of tension at 60-70 % epiboly (p = 0.0245) between the E-YSL and the vegetal pole (right). Standard Errors are displayed.


( a ) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut ( red line time point ) in the E - YSL of a Myosin - GFP transgenic embryo [ Tg ( β - actin : myl 12 . 1 - e - GFP ) ] . After a rapid opening , an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched ( elastic ) than the vegetal yolk cellmembrane / cortex . When the gap is closed , the cut center returns to its initial position and the elastic equilibrium is restored . Scale bar 10 μm .

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(a) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut (red line time point) in the E-YSL of a Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. After a rapid opening, an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched (elastic) than the vegetal yolk cellmembrane/cortex. When the gap is closed, the cut center returns to its initial position and the elastic equilibrium is restored. Scale bar 10 μm.


( d ) Dynamic simulation of AV and CC stresses normalized values in arbitrary units ( from Movie EV9 - 40 % ; 65 % ; 80 % epiboly ) represented as in Figure 1a . The AV and CC stresses are equal at the poles , as the epiboly simulation progresses , the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin .

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(d) Dynamic simulation of AV and CC stresses normalized values in arbitrary units (from Movie EV9 - 40 %; 65 %; 80 % epiboly) represented as in Figure 1a. The AV and CC stresses are equal at the poles, as the epiboly simulation progresses, the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin.


( a ) Speed of the EVL advancing front at different epiboly stages ( φ angle ) for two distinct simulated conditions : 1 ) a difference of elastic tension between the EVL and the yolk associated to a two - phase reduction of the contractile E - YSL width ( from 20º to 5º ) as epiboly advances ( from 50º to 155º ) ( wild type - blue ) . The contractile E - YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2 . 25 times faster , and 2 ) Same assumptions as in 1 but with no shrinkage of the E - YSL ( constant 20º ) ( contractileless - red ) .

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(a) Speed of the EVL advancing front at different epiboly stages (φ angle) for two distinct simulated conditions: 1) a difference of elastic tension between the EVL and the yolk associated to a two-phase reduction of the contractile E-YSL width (from 20º to 5º) as epiboly advances (from 50º to 155º) (wild type - blue). The contractile E-YSL width decreases at a slower rate until 80º and shrinks afterwards at a rate 2.25 times faster, and 2) Same assumptions as in 1 but with no shrinkage of the E-YSL (constant 20º) (contractileless - red).


( b ) Experimental values for the EVL advancing front speeds at different epiboly stages ( φ angle - top ) and at different times ( bottom ) for wild type controls ( blue ) and Msn1yolk morphants with reduced E - YSL contractility ( red ) . Experimental profiles closely approach to those inferred in the simulations ( compare to ( a ) ) .

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(b) Experimental values for the EVL advancing front speeds at different epiboly stages (φ angle - top) and at different times (bottom) for wild type controls (blue) and Msn1yolk morphants with reduced E-YSL contractility (red). Experimental profiles closely approach to those inferred in the simulations (compare to (a)).


( c ) Timeline showing medial sections and power isolines for wild type ( top ) and Msn1yolk morphants ( bottom ) along epiboly . Mechanical parameters were inferred by HR from experimental velocity fields ( Movie EV10 ) . The RMSE of power is shown as a percentage at each time point . DCs in the Msn1yolk morphants undergo ingression on schedule at 50 % epiboly ( yellow arrow ) . Importantly , as in wild type controls , the active work in morphants is detected at the E - YSL ( red arrow ) . This power supply fades away as epiboly slows down and eventually fails . Scale bar 100 μm .

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(c) Timeline showing medial sections and power isolines for wild type (top) and Msn1yolk morphants (bottom) along epiboly. Mechanical parameters were inferred by HR from experimental velocity fields (Movie EV10). The RMSE of power is shown as a percentage at each time point. DCs in the Msn1yolk morphants undergo ingression on schedule at 50 % epiboly (yellow arrow). Importantly, as in wild type controls, the active work in morphants is detected at the E-YSL (red arrow). This power supply fades away as epiboly slows down and eventually fails. Scale bar 100 μm.


( d ) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent ( 9 hours after egg laying ) for Msn1yolk morphants . The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants .

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(d) Tensional profiles normalized values in arbitrary units inferred by HR for wild type at 80 % epiboly and its temporal equivalent (9 hours after egg laying) for Msn1yolk morphants. The characteristic profiles of longitudinal and latitudinal stresses and the animal to vegetal stress gradient that are built up during epiboly are never properly developed in morphants.


A The expression of LINC00839 in CRC tissues and normal mucosa tissues in the UALCAN database . B , C , D The expression of LINC00839 in unpaired CRC and normal samples in the LnCAR database ( GSE37364 , GSE71187 , and GSE33113 ) . Data information : In A , B , C , D middle lines represent the median , and the upper and lower lines represent the upper and lower quartiles . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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A The expression of LINC00839 in CRC tissues and normal mucosa tissues in the UALCAN database. B, C, D The expression of LINC00839 in unpaired CRC and normal samples in the LnCAR database (GSE37364, GSE71187, and GSE33113). Data information: In A, B, C, D middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


E , F The expression levels of LINC00839 in tumour tissues and NATs from the CRC cohort ( n = 45 ) . nmCRC denotes CRC patients without metastases , and mCRC denotes CRC patients with metastases . Data information : In E , F , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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E, F The expression levels of LINC00839 in tumour tissues and NATs from the CRC cohort (n = 45). nmCRC denotes CRC patients without metastases, and mCRC denotes CRC patients with metastases. Data information: In E, F, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


G Representative ISH staining of LINC00839 in paraffin - embedded samples of CRC tumour tissues and NATs . ( a ) Expression of LINC00839 in tumour tissues and NATs . Scale bars , 100 μm . ( b ) Magnification of normal mucosal area and ( c ) magnification of the tumour area . Scale bars , 50 μm . ( d ) Weakly positive staining of LINC00839 in normal mucosa . Scale bars , 50 μm . ( e ) Strong positive staining of LINC00839 in tumours and ( f ) magnification of the local area . Scale bars , 100 μm .

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G Representative ISH staining of LINC00839 in paraffin-embedded samples of CRC tumour tissues and NATs. (a) Expression of LINC00839 in tumour tissues and NATs. Scale bars, 100 μm. (b) Magnification of normal mucosal area and (c) magnification of the tumour area. Scale bars, 50 μm. (d) Weakly positive staining of LINC00839 in normal mucosa. Scale bars, 50 μm. (e) Strong positive staining of LINC00839 in tumours and (f) magnification of the local area. Scale bars, 100 μm.


H The expression score of the tumours and NATs in the ISH cohort ( n = 222 ) . Data information : In H the data are shown as the mean ± SD and were analysed by Student ' s t test . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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H The expression score of the tumours and NATs in the ISH cohort (n = 222). Data information: In H the data are shown as the mean ± SD and were analysed by Student's t test. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


I Differential expression of LINC00839 in nonmetastatic CRC patients ( nmCRC ) and metastatic CRC patients ( mCRC ) . Data information : In I , the data are shown as the mean ± SD and were analysed by Student ' s t test . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

I Differential expression of LINC00839 in nonmetastatic CRC patients (nmCRC) and metastatic CRC patients (mCRC). Data information: In I, the data are shown as the mean ± SD and were analysed by Student's t test. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


J Expression of LINC00839 in CRC patients with early - stage ( stage I and II ) and advanced - stage ( stage III and IV ) disease . Data information : In J , middle lines represent the median , and the upper and lower lines represent the upper and lower quartiles . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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J Expression of LINC00839 in CRC patients with early-stage (stage I and II) and advanced-stage (stage III and IV) disease. Data information: In J, middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


Kaplan - Meier analysis of overall survival rate of the ISH CRC cohort .

Kaplan-Meier analysis of overall survival rate of the ISH CRC cohort.


L Kaplan - Meier analysis of overall disease - free survival rate of the ISH CRC cohort . M Kaplan - Meier analysis of the disease - free survival rate of CRC patients in the LnCAR database ( GSE33113 ) .

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L Kaplan-Meier analysis of overall disease-free survival rate of the ISH CRC cohort. M Kaplan-Meier analysis of the disease-free survival rate of CRC patients in the LnCAR database (GSE33113).


C Colony formation assay was performed with LINC00839 - overexpressing cells and vector control cells . , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 biological replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Colony formation assay was performed with LINC00839-overexpressing cells and vector control cells. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


D Wound - healing assay was performed with LINC00839 - overexpressing cells and vector control cells . Scale bars , 100 μm . , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 biological replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Wound-healing assay was performed with LINC00839-overexpressing cells and vector control cells. Scale bars, 100 μm. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


E Matrigel invasion assay was performed with LINC00839 - overexpressing cells . Scale bars , 100 μm . , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 biological replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Matrigel invasion assay was performed with LINC00839-overexpressing cells. Scale bars, 100 μm. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


F The CDX model showing the effect of LINC00839 on tumour growth . Representative image of tumours harvested from the mouse model ( upper panel ) , the quantitative analysis of tumour weight and tumour volume ( medium panel ) , and representative images and quantification of IHC staining of Ki - 67 in tumours ( bottom panel ) ( n = 6 for each cohort , and the results are presented as the mean ± SD , Student ' s t test ( medium left and low right panel ) and ANOVA ( medium right ) . Scale bars , 100 μm ( n = 6 biological replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CDX model showing the effect of LINC00839 on tumour growth. Representative image of tumours harvested from the mouse model (upper panel), the quantitative analysis of tumour weight and tumour volume (medium panel), and representative images and quantification of IHC staining of Ki-67 in tumours (bottom panel) (n = 6 for each cohort, and the results are presented as the mean ± SD, Student's t test (medium left and low right panel) and ANOVA (medium right). Scale bars, 100 μm (n = 6 biological replicates . Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


. G The CRC orthotopic mouse model revealed the effect of LINC00839 on tumour development . Representative images of primary tumours ( yellow arrowheads ) and liver metastasis ( blue arrowheads ) in the model ( upper panel ) , analysis of tumour volume and the number of mice with liver metastasis ( medium panel ) , and H & E staining of tumours and metastases ( n = 6 for each cohort ) . Scale bars , 100 μm ( n = 6 biological replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The CRC orthotopic mouse model revealed the effect of LINC00839 on tumour development. Representative images of primary tumours (yellow arrowheads) and liver metastasis (blue arrowheads) in the model (upper panel), analysis of tumour volume and the number of mice with liver metastasis (medium panel), and H&E staining of tumours and metastases (n = 6 for each cohort). Scale bars, 100 μm (n = 6 biological replicates . Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


A An RNA pull - down assay was performed to identify proteins that bind to LINC00839 in HCT116 cells .

A An RNA pull-down assay was performed to identify proteins that bind to LINC00839 in HCT116 cells.


B RNA pull - down assay and WB showed that biotinylated LINC00839 can bind to Ruvb1 in HCT116 cells . Dot blot of RNA - protein interactions indicating equal amounts of RNA used in the 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

B RNA pull-down assay and WB showed that biotinylated LINC00839 can bind to Ruvb1 in HCT116 cells. Dot blot of RNA-protein interactions indicating equal amounts of RNA used in the assay.


C RIP experiments were performed to confirm the binding of LINC00839 to Ruvb1 . IgG was used as a negative control antibody . Primers specific for U6 and GAPDH were used as negative control primers . Data information : In C , the data are presented as the mean ± SD and were analysed by unpaired t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RIP experiments were performed to confirm the binding of LINC00839 to Ruvb1. IgG was used as a negative control antibody. Primers specific for U6 and GAPDH were used as negative control primers. Data information: In C, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


D Different fragments of LINC00839 ( solid line ) and its antisense sequence ( dotted line ) were used for the RNA pull - down assay and the subsequent WB .

O O O O O O O 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 Different fragments of LINC00839 (solid line) and its antisense sequence (dotted line) were used for the RNA pull-down assay and the subsequent WB.


E Full length LINC00839 , antisense sequence of LINC00839 , and LINC00839 sequence lacking nucleotides 1033 - 1290 were used for the RNA pull - down assay and the subsequent WB .

O O O O O O O O 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 Full length LINC00839, antisense sequence of LINC00839, and LINC00839 sequence lacking nucleotides 1033-1290 were used for the RNA pull-down assay and the subsequent WB.


F RIP experiments were performed by incubating Tip60 , INO80 , SRCAP , and PIH1D1 antibodies with HCT116 cell lysates . Data information : In F , the data are presented as the mean ± SD and were analysed by unpaired t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RIP experiments were performed by incubating Tip60, INO80, SRCAP, and PIH1D1 antibodies with HCT116 cell lysates. Data information: In F, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


G The effect of LINC00839 on the expression of Tip60 and Ruvb1 was determined by WB .

G The effect of LINC00839 on the expression of Tip60 and Ruvb1 was determined by WB.


H Interaction of endogenous Ruvb1 and Tip60 in CRC cells was confirmed by Co - IP .

H Interaction of endogenous Ruvb1 and Tip60 in CRC cells was confirmed by Co-IP.


I The interaction between Ruvb1 and Tip60 in cells overexpressing wild - type LINC00839 and LINC - Δ5 was confirmed by Co - IP .

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

I The interaction between Ruvb1 and Tip60 in cells overexpressing wild-type LINC00839 and LINC-Δ5 was confirmed by Co-IP.


J The interaction between Ruvb1 and Tip60 in LINC00839 - KD cells and cells expressing LINC - Δ5 was confirmed by Co - IP .

J The interaction between Ruvb1 and Tip60 in LINC00839-KD cells and cells expressing LINC-Δ5 was confirmed by Co-IP.


K Acetylase activity assays were performed with LINC00839 - overexpressing and LINC00839 - knockdown cells . Data information : In the data are presented as the mean ± SD and were analysed by unpaired t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

K Acetylase activity assays were performed with LINC00839-overexpressing and LINC00839-knockdown cells. Data information: In the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


L Acetylase activity was measured in SW480 and LoVo cells subjected to different treatments . Data information : In L , the data are presented as the mean ± SD and were analysed by unpaired t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

L Acetylase activity was measured in SW480 and LoVo cells subjected to different treatments. Data information: In L, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


B Heatmap of DEGs related to OXPHOS and mitochondrial biogenesis . Expression levels are shown in log2 scale of normalized intensity values ( see colour gradient ) ranging from low ( blue ) to high ( red ) expression .

O O O O O O O O O O O O O O O O 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 Heatmap of DEGs related to OXPHOS and mitochondrial biogenesis. Expression levels are shown in log2 scale of normalized intensity values (see colour gradient) ranging from low (blue) to high (red) expression.


D , E The ATP levels and the NAD / NADH ratio were measured in LINC00839 - overexpressing and LINC00839 - KD cells ( mean ± SD , Student ' s t test , n = 3 biological replicates ) . Data information : Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E The ATP levels and the NAD/NADH ratio were measured in LINC00839-overexpressing and LINC00839-KD cells (mean ± SD, Student's t test, n = 3 biological replicates). Data information: Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


F Expression of MT - ND5 , MT - CYB , MT - CO1 , and TFAM in LINC00839 - overexpressing and LINC00839 - KD cells as determined by WB .

F Expression of MT-ND5, MT-CYB, MT-CO1, and TFAM in LINC00839-overexpressing and LINC00839-KD cells as determined by WB.


G Analysis of NRF1 expression in CRC tissues ( T ) and normal tissues ( N ) the GEPIA database . Data information : In G , the middle lines represent the median , and the upper and lower lines represent the upper and lower quartiles ; the data were analysed by ANOVA . Across experiments , the P values are as follows : ns = not significant , * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Analysis of NRF1 expression in CRC tissues (T) and normal tissues (N) the GEPIA database. Data information: In G, the middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles; the data were analysed by ANOVA. Across experiments, the P values are as follows: ns = not significant, *P < 0.05


H Correlation analysis of LINC00839 and NRF1 mRNA levels in the TCGA and GTEx databases ( Pearson ) .

H Correlation analysis of LINC00839 and NRF1 mRNA levels in the TCGA and GTEx databases (Pearson).


I , Expression of NRF1 in LINC00839 - overexpressin and LINC00839 - KD cells was measured by qPCR Data information : In I , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

I, Expression of NRF1 in LINC00839-overexpressin and LINC00839-KD cells was measured by qPCR Data information: In I, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


J Expression of NRF1 in LINC00839 - overexpressin and LINC00839 - KD cells was measured by WB .

J Expression of NRF1 in LINC00839-overexpressin and LINC00839-KD cells was measured by WB.


K , L The enrichment of the NRF1 promoter ( P1 - P10 ) by H4K5ac and H4K8ac antibodies in LINC00839 - overexpressing and control LoVo cells as determined by CHIP assay . Normal IgG was used as a negative control . Data information : In K , L , , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

K, L The enrichment of the NRF1 promoter (P1-P10) by H4K5ac and H4K8ac antibodies in LINC00839-overexpressing and control LoVo cells as determined by CHIP assay. Normal IgG was used as a negative control. Data information: In K, L, , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


M , N The enrichment of the P2 and P3 fragments by H4K5ac and H4K8ac antibodies in LoVo cells as determined by CHIP assay . Normal IgG was used as a negative control . Data information : In M , N , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

M, N The enrichment of the P2 and P3 fragments by H4K5ac and H4K8ac antibodies in LoVo cells as determined by CHIP assay. Normal IgG was used as a negative control. Data information: In M, N , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


O The enrichment of NRF1 with a probe targeting LINC00839 relative to a NC probe in HCT116 cells , as determined by ChIRP assay . LacZ served as a negative control ( NC ) . Data information : In O , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 technical replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

O The enrichment of NRF1 with a probe targeting LINC00839 relative to a NC probe in HCT116 cells, as determined by ChIRP assay. LacZ served as a negative control (NC). Data information: In O, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


A , B ATP levels and NAD / NADH ratios were measured in SW480 and LoVo cells . Data information : In A , B , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 biological replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B ATP levels and NAD/NADH ratios were measured in SW480 and LoVo cells. Data information: In A, B, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


C Expression of genes related to mitochondrial biogenesis and EMT in SW480 and LoVo cells as determined by WB .

C Expression of genes related to mitochondrial biogenesis and EMT in SW480 and LoVo cells as determined by WB.


D Invasion of SW480 and LoVo cells was assessed by Matrigel invasion assay . Scale bars , 100 μm . Data information : In D , the data are presented as the mean ± SD and were analysed by Student ' s t test , n = 3 biological replicates . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Invasion of SW480 and LoVo cells was assessed by Matrigel invasion assay. Scale bars, 100 μm. Data information: In D, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


E Representative images of primary tumours ( yellow arrowhead ) and metastases in the orthotopic implantation models ( n = 6 for each cohort , left panel ) . The number of mice with liver metastasis ( blue arrowhead ) was determined . The primary tumour volume in each group was calculated ( right panel ) . H & E staining of tumours and metastases ( bottom panel ) . Scale bars , 200 μm ( n = 6 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of primary tumours (yellow arrowhead) and metastases in the orthotopic implantation models (n = 6 for each cohort, left panel). The number of mice with liver metastasis (blue arrowhead) was determined. The primary tumour volume in each group was calculated (right panel). H&E staining of tumours and metastases (bottom panel). Scale bars, 200 μm (n = 6 biological replicates).


F ISH staining of LINC00839 and IHC staining of NRF1 , H4K5ac , and H4K8ac in paraffin - embedded samples of CRC tumour tissues and NATs . Scale bars , 200 μm .

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

F ISH staining of LINC00839 and IHC staining of NRF1, H4K5ac, and H4K8ac in paraffin-embedded samples of CRC tumour tissues and NATs. Scale bars, 200 μm.


G , H , Correlation analysis of the expression of LINC00839 and NRF1 ( G ) , H4K5ac ( H ) in CRC patients ( n = 120 ) . Data information : In G , H show the Pearson correlation coefficient results . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

G, H, Correlation analysis of the expression of LINC00839 and NRF1 (G), H4K5ac (H) in CRC patients (n = 120). Data information: In G, H show the Pearson correlation coefficient results. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


I Correlation analysis of the expression of LINC00839 and H4K8ac ( I ) in CRC patients ( n = 120 ) . Data information : In I show the Pearson correlation coefficient results . Across experiments , the P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

I Correlation analysis of the expression of LINC00839 and H4K8ac (I) in CRC patients (n = 120). Data information: In I show the Pearson correlation coefficient results. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


J Correlation of NRF1 expression with the overall survival rate in patients with CRC ( n = 120 ) . NRF1 expression was stratified as high and low according to the median expression level .

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J Correlation of NRF1 expression with the overall survival rate in patients with CRC (n = 120). NRF1 expression was stratified as high and low according to the median expression level.


( a ) MDCK / pGFP - LC3 cells infected with a wild - type or ΔactA2 strain of L . monocytogenes were stained with an anti - Listeria antibody ( red ) and rhodamine phalloidin ( stains F - actin ; light blue ) . Scale bars , 10 μm . Arrowheads indicate GFP - LC3 - positive bacteria . ( b ) High magnification of framed images in a , and graphs show intensity of the fluoresence signal along the arrow . ( c ) Quantification of the number of GFP - LC3 - positive bacteria shown in a . Data are mean ± s . e . m . At least 500 bacteria were counted in each experiment ( n = 7 and 6 experiments at 2 and 4 h , respectively ) .

(a) MDCK/pGFP-LC3 cells infected with a wild-type or ΔactA2 strain of L. monocytogenes were stained with an anti-Listeria antibody (red) and rhodamine phalloidin (stains F-actin; light blue). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive bacteria. (b) High magnification of framed images in a, and graphs show intensity of the fluoresence signal along the arrow. (c) Quantification of the number of GFP-LC3-positive bacteria shown in a. Data are mean ± s.e.m. At least 500 bacteria were counted in each experiment (n = 7 and 6 experiments at 2 and 4 h, respectively).


( d ) LC3 levels in infected MDCK cells . MDCK cells were infected with wild - type or ΔactA2 bacteria for the indicated times . EBSS ( Earle ' s balanced salt solution , a starvation medium ) and rapamycin ( 100 μg ml − 1 ) were used as controls .

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(d) LC3 levels in infected MDCK cells. MDCK cells were infected with wild-type or ΔactA2 bacteria for the indicated times. EBSS (Earle's balanced salt solution, a starvation medium) and rapamycin (100 μg ml−1) were used as controls.


( e - f ) Intracellular survival of wild - type or ΔactA2 bacteria in MDCK cells ( e ; n = 8 ) , MDCK cells treated with 10 mM 3 - MA ( f ; n = 4 ) ,

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(e-f) Intracellular survival of wild-type or ΔactA2 bacteria in MDCK cells (e; n = 8), MDCK cells treated with 10 mM 3-MA (f; n = 4),


( e - h ) Intracellular survival of wild - type or ΔactA2 bacteria in atg5 + / + MEF cells ( g ; n = 8 and 7 for wild - type and ΔactA2 , respectively ) or atg5 − / − MEF cells ( h ; n = 4 and 3 for wild - type and ΔactA2 , respectively ) . Data are presented as the number of colony - forming units ( CFU ) relative to that at 30 min after infection , and are mean ± s . e . m . WT , wild type .

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(e-h) Intracellular survival of wild-type or ΔactA2 bacteria in atg5+/+ MEF cells (g; n = 8 and 7 for wild-type and ΔactA2, respectively) or atg5−/− MEF cells (h; n = 4 and 3 for wild-type and ΔactA2, respectively). Data are presented as the number of colony-forming units (CFU) relative to that at 30 min after infection, and are mean ± s.e.m. WT, wild type.


( b , c ) Quantification of the number of GFP - LC3 - positive bacteria ( b ) or actin - accumulating bacteria ( c ) . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 3 experiments ) .

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(b, c) Quantification of the number of GFP-LC3-positive bacteria (b) or actin-accumulating bacteria (c). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments).


( d ) Colocalization of ubiquitin with ΔactA2 . MDCK / pGFP - LC3 cells infected with ΔactA2 bacteria were stained with an anti - Listeria antibody ( blue ) and anti - ubiquitin antibody ( red ) . Scale bars , 10 μm . Arrowheads indicate GFP - LC3 - positive or ubiquitin - positive bacteria . ( e ) High magnification of framed image in d , and the graph shows the intensity of the fluorescence signal along the arrow . ( f ) Quantification of the number of GFP - LC3 - positive , ubiquitin - positive or GFP - LC3 - and ubiquitin - positive bacteria . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 3 experiments ) .

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(d) Colocalization of ubiquitin with ΔactA2. MDCK/pGFP-LC3 cells infected with ΔactA2 bacteria were stained with an anti-Listeria antibody (blue) and anti-ubiquitin antibody (red). Scale bars, 10 μm. Arrowheads indicate GFP-LC3-positive or ubiquitin-positive bacteria. (e) High magnification of framed image in d, and the graph shows the intensity of the fluorescence signal along the arrow. (f) Quantification of the number of GFP-LC3-positive, ubiquitin-positive or GFP-LC3- and ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments).


( g ) Immunogold electron micrograph of MDCK / pGFP - LC3 cells infected with ΔactA2 . Gold particles of 5 nm ( arrowheads ) indicate GFP - LC3 , and gold particles of 10 nm ( arrows ) indicate ubiquitin . Scale bars , 0 . 5 μm .

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(g) Immunogold electron micrograph of MDCK/pGFP-LC3 cells infected with ΔactA2. Gold particles of 5 nm (arrowheads) indicate GFP-LC3, and gold particles of 10 nm (arrows) indicate ubiquitin. Scale bars, 0.5 μm.


( h ) Kinetics of the generation of GFP - LC3 - positive or ubiquitin - positive bacteria . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 3 experiments ) . * P 0 . 001 . ( i ) Quantification of the number of ubiquitin - positive or GFP - LC3 - positive bacteria . MDCK / pGFP - LC3 cells infected with wild - type or ΔactA2 bacteria were untreated or treated with 3 - MA ( 10 mM ) . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n ≥ 3 experiments ) . Ub , ubiquitin . WT , wild type .

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(h) Kinetics of the generation of GFP-LC3-positive or ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). *P 0.001. (i) Quantification of the number of ubiquitin-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3 cells infected with wild-type or ΔactA2 bacteria were untreated or treated with 3-MA (10 mM). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n ≥ 3 experiments). Ub, ubiquitin. WT, wild type.


( a ) Quantification of the number of ubiquitin - positive , p62 - positive or GFP - LC3 - positive bacteria . MDCK / pGFP - LC3 / pp62 - 3 × Myc cells were infected with a wild - type or ΔactA2 strain of L . monocytogenes for 2 h . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 6 experiments ) .

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(a) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. MDCK/pGFP-LC3/pp62-3×Myc cells were infected with a wild-type or ΔactA2 strain of L. monocytogenes for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 experiments).


( b ) Colocalization of p62 with ΔactA2 . MDCK / pGFP - LC3 / pp62 - 3 × Myc cells were stained with an anti - ubiquitin antibody ( light blue ) , anti - Myc antibody ( red ) , DAPI ( white ) and anti - GFP - LC3 ( green ) . Scale bar , 10 μm . Arrowheads indicate GFP - LC3 - , ubiquitin - or p62 - positive bacteria .

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(b) Colocalization of p62 with ΔactA2. MDCK/pGFP-LC3/pp62-3×Myc cells were stained with an anti-ubiquitin antibody (light blue), anti-Myc antibody (red), DAPI (white) and anti-GFP-LC3 (green). Scale bar, 10 μm. Arrowheads indicate GFP-LC3-, ubiquitin- or p62-positive bacteria.


( c ) Quantification of the number of ubiquitin - positive , p62 - positive or GFP - LC3 - positive bacteria . p62 − / − / p62 cells were infected with ΔactA2 bacteria for 2 h . Data are mean ± s . e . m . , at least 500 bacteria were counted in each experiment ( n = 6 and 3 experiments for p62 − / − and p62 − / − / p62 , respectively )

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(c) Quantification of the number of ubiquitin-positive, p62-positive or GFP-LC3-positive bacteria. p62−/−/p62 cells were infected with ΔactA2 bacteria for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 and 3 experiments for p62−/− and p62−/−/p62, respectively)


( d , e ) Intracellular survival of wild - type or ΔactA2 bacteria in p62 − / − cells ( d ) or p62 − / − / p62 cells ( e ; colony - forming units , CFU , relative to CFU 30 min after infection ) . Data are presented as mean ± s . e . m . ( n ≥ 4 experiments ) .

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(d, e) Intracellular survival of wild-type or ΔactA2 bacteria in p62−/− cells (d) or p62−/−/p62 cells (e; colony-forming units, CFU, relative to CFU 30 min after infection). Data are presented as mean ± s.e.m. (n ≥ 4 experiments).


( g ) Generation of p62 - 3 × Myc mutants in p62 − / − cells .

(g) Generation of p62-3×Myc mutants in p62−/− cells.


( h ) Quantification of the number of GFP - positive or GFP - LC3 - positive bacteria . p62 − / − / p62 ( wild type , ΔLIR , ΔUBA , ΔLIR - ΔUBA or K7AD69A ) cells were infected with ΔactA2 bacteria and stained with anti - bacteria and anti - Myc antibodies ( Supplementary Information , Fig . S4e ) . Data are mean ± s . e . m . , at least 500 LC3 were counted in each experiment ( n = 3 experiments ) . P 0 . 001 versus p62 - positive LC3 in p62 − / − / p62 wild - type cells , P 0 . 001 versus GFP - p62 - positive bacteria in p62 − / − / ubiquitinwild - type cells . Ub , ubiquitin . WT , wild type . A full scan of g is shown in Supplementary Information , Fig . S7 .

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(h) Quantification of the number of GFP-positive or GFP-LC3-positive bacteria. p62−/−/p62 (wild type, ΔLIR, ΔUBA, ΔLIR-ΔUBA or K7AD69A) cells were infected with ΔactA2 bacteria and stained with anti-bacteria and anti-Myc antibodies (Supplementary Information, Fig. S4e). Data are mean ± s.e.m., at least 500 LC3 were counted in each experiment (n = 3 experiments). P 0.001 versus p62-positive LC3 in p62−/−/p62 wild-type cells, P 0.001 versus GFP-p62-positive bacteria in p62−/−/ubiquitinwild-type cells. Ub, ubiquitin. WT, wild type. A full scan of g is shown in Supplementary Information, Fig. S7.


( b - e ) The series of pEGFP - ActA - Q79C constructs was transfected into COS - 7 cells . Cells were fixed with 4 % Paraformaldehyde after 18 h and stained with an anti - Arp2 antibody ( b ) , an anti - VASP antibody ( c ) , an anti - ubiquitin antibody ( d ) or an anti - p62 antibody ( e ; red ) . Scale bars , 10 μm . Arrowheads indicate Arp2 - , VASP - , ubiquitin - or p62 - positive Q79C . Ub , ubiquitin .

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(b-e) The series of pEGFP-ActA-Q79C constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 18 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive Q79C. Ub, ubiquitin.


( b - e ) The series of pEGFP - ActA - 170 constructs was transfected into COS - 7 cells . Cells were fixed with 4 % Paraformaldehyde after 14 h and stained with an anti - Arp2 antibody ( b ) , an anti - VASP antibody ( c ) , an anti - ubiquitin antibody ( d ) or an anti - p62 antibody ( e ; red ) . Scale bars , 10 μm . Arrowheads indicate Arp2 - , VASP - , ubiquitin - or p62 - positive 170 . Ub , ubiquitin .

(b-e) The series of pEGFP-ActA-170 constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 14 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive 170. Ub, ubiquitin.


A , B Virus titers in the blood from mice ( n = 5 ) fed an NSD ( 0 . 45 % NaCl ) or HSD ( 4 % NaCl ) for 30 days ( A ) or 7 days ( B ) and then intraperitoneally infected with VSV ( 1x108 PFU per gram body , 48 hrs ) were analyzed by the TCID50 assay . Data information : Data represent mean and SEM of five biological replicates For all statistical testing , p values were calculated using two - tailed unpaired Student ' s t - test . NS , not significant ( p > 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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A, B Virus titers in the blood from mice (n=5) fed an NSD (0.45% NaCl) or HSD (4% NaCl) for 30 days (A) or 7 days (B) and then intraperitoneally infected with VSV (1x108 PFU per gram body, 48 hrs) were analyzed by the TCID50 assay. Data information: Data represent mean and SEM of five biological replicates For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.


H RT - qPCR analysis of viral RNA levels in U937 , THP1 , HT1080 , 2fTGH and A549 cells infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . I RAW264 . 7 cells were infected with VSV / H1N1 / SeV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) , or were transfected with HBV - replicon or HCV - replicon and then treated with additional NaCl ( + 34 mM ) for 12 hrs . Viral RNA levels were analyzed by RT - qPCR . Data information : Data show the mean and SD of four or three biological replicates . For all statistical testing , p values were calculated using two - tailed unpaired Student ' s t - test . NS , not significant ( p > 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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H RT-qPCR analysis of viral RNA levels in U937, THP1, HT1080, 2fTGH and A549 cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). I RAW264.7 cells were infected with VSV/H1N1/SeV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM), or were transfected with HBV-replicon or HCV-replicon and then treated with additional NaCl (+34 mM) for 12 hrs. Viral RNA levels were analyzed by RT-qPCR. Data information: Data show the mean and SD of four or three biological replicates. For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.


A Western blot analysis of VSV - G protein levels in Ifnar1 + / + or Ifnar1 - / - MEF cells infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : are representative of at least two biological

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A Western blot analysis of VSV-G protein levels in Ifnar1+/+ or Ifnar1-/- MEF cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: are representative of at least two biological replicates


C RAW264 . 7 cells were treated with mIFNβ ( 50 IU / ml , 20 hrs ) immediately after addition of NaCl ( + 17 , 34 and 51 mM ) . After washing , cells were infected with H1N1 ( MOI = 1 . 0 , 24 hrs ) and viral RNA levels were analyzed by RT - qPCR . Data information : Data represent mean and SD of three biological replicates , and p values were calculated using two - tailed unpaired Student ' s t - test For all statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) , * * p < 0 . 01 and * * * p < 0 . 001 .

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C RAW264.7 cells were treated with mIFNβ (50 IU/ml, 20 hrs) immediately after addition of NaCl (+17, 34 and 51 mM). After washing, cells were infected with H1N1 (MOI=1.0, 24 hrs) and viral RNA levels were analyzed by RT-qPCR. Data information: Data represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not signiﬁcant (p > 0.05), **p < 0.01 and ***p < 0.001.


F RT - qPCR analysis of Ifit1 , Isg54 and Isg15 mRNA levels in HEK293T pretreated with additional NaCl ( + 34 mM ) for 12 hrs and then treated with IFNα ( 1 , 000 IU / ml ) as indicated . Data information : Data represent mean and SD of three biological replicates , and p values were calculated using two - tailed unpaired Student ' s t - test For all statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) , * * p < 0 . 01 and * * * p < 0 . 001 .

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F RT-qPCR analysis of Ifit1, Isg54 and Isg15 mRNA levels in HEK293T pretreated with additional NaCl (+34 mM) for 12 hrs and then treated with IFNα (1,000 IU/ml) as indicated. Data information: Data represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not signiﬁcant (p > 0.05), **p < 0.01 and ***p < 0.001.


G RAW264 . 7 cells were treated with mIFNβ ( 500 IU / ml ) for 12 hrs immediately after addition of NaCl ( + 34 mM ) or control ddH2O ( Ctrl ) . Whole cell lysates were subjected to a quantitative proteomic analysis with a TMT2 - plex method to identify the differentially expressed proteins . The upregulated and downregulated proteins were analyzed in the volcano plot . The most dramatically downregulated ISG protein , Viperin ( Rsad2 ) , was marked in the dotted frame .

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G RAW264.7 cells were treated with mIFNβ (500 IU/ml) for 12 hrs immediately after addition of NaCl (+34 mM) or control ddH2O (Ctrl). Whole cell lysates were subjected to a quantitative proteomic analysis with a TMT2-plex method to identify the differentially expressed proteins. The upregulated and downregulated proteins were analyzed in the volcano plot. The most dramatically downregulated ISG protein, Viperin (Rsad2), was marked in the dotted frame.


H Western blot analysis of Viperin protein in RAW264 . 7 treated with mIFNβ ( 100 and 500 IU / ml , 12 hrs ) immediately after addition of NaCl ( + 34 mM ) . I Western blot analysis of Viperin protein in HT1080 treated with IFNα ( 1 , 000 IU / ml ) and NaCl ( + 34 mM ) as ( H ) . J Western blot analysis of Viperin protein in MEF cells infected with VSV ( MOI = 0 . 5 and 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . K Western blot analysis of Viperin protein in THP1 infected with H1N1 ( MOI = 1 . 0 ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data are representative of at least two biological

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H Western blot analysis of Viperin protein in RAW264.7 treated with mIFNβ (100 and 500 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). I Western blot analysis of Viperin protein in HT1080 treated with IFNα (1,000 IU/ml) and NaCl (+34 mM) as (H). J Western blot analysis of Viperin protein in MEF cells infected with VSV (MOI=0.5 and 1.0, 24 hrs) immediately after addition of NaCl (+34 mM). K Western blot analysis of Viperin protein in THP1 infected with H1N1 (MOI= 1.0) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates


N Survival curves of 8 - week - old Rsad2 + / + or Rsad2 - / - mice administrated with an NSD or HSD for 7 days and then intraperitoneally infected with VSV ( 1 × 108 PFU per gram body mouse , n = 10 ) . Data information : Data ( N ) show the mean and SEM of ten biological replicates , and p value was calculated using logrank ( Mantel - Cox ) tests . For all statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) , * * p < 0 . 01 and * * * p < 0 . 001 .

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N Survival curves of 8-week-old Rsad2+/+ or Rsad2-/- mice administrated with an NSD or HSD for 7 days and then intraperitoneally infected with VSV (1×108 PFU per gram body mouse, n=10). Data information: Data (N) show the mean and SEM of ten biological replicates, and p value was calculated using logrank (Mantel-Cox) tests. For all statistical testing: NS, not signiﬁcant (p > 0.05), **p < 0.01 and ***p < 0.001.


B Western blot analysis of FH - Viperin protein in stable FH - Viperin - expressing HeLa cells pretreated with additional NaCl ( + 34 mM ) and then treated with CHX ( 50 μM ) for 6 and 12 hrs . Data information : Data are representative of at least two biological replicates .

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B Western blot analysis of FH-Viperin protein in stable FH-Viperin-expressing HeLa cells pretreated with additional NaCl (+34 mM) and then treated with CHX (50 μM) for 6 and 12 hrs. Data information: Data are representative of at least two biological replicates.


C RAW264 . 7 were treated with MG132 ( 10 μM ) and additional NaCl ( + 34 mM ) , followed by mIFNβ ( 300 IU / ml ) treatment for 12 hrs . Viperin protein levels were analyzed by immunoblotting . Data information : Data are representative of at least two biological replicates .

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C RAW264.7 were treated with MG132 (10 μM) and additional NaCl (+34 mM), followed by mIFNβ (300 IU/ml) treatment for 12 hrs. Viperin protein levels were analyzed by immunoblotting. Data information: Data are representative of at least two biological replicates.


J Western blot analysis of Viperin in 2fTGH cells transfected with control shRNAs ( shCtrl ) or shRNAs against USP33 ( shUSP33 ) and then treated with IFNα ( 1 , 000 IU / ml ) for different times . Data information : Data are representative of at least two biological replicates .

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

J Western blot analysis of Viperin in 2fTGH cells transfected with control shRNAs (shCtrl) or shRNAs against USP33 (shUSP33) and then treated with IFNα (1,000 IU/ml) for different times. Data information: Data are representative of at least two biological replicates.


M Western blot analysis of Viperin protein in Usp33 - / - MEF cells transfected with FH - USP33 ( wild type , WT ; C194S and H673Q , DM ) and then stimulated with mIFNβ ( 500 IU / ml ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

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

M Western blot analysis of Viperin protein in Usp33-/- MEF cells transfected with FH-USP33 (wild type, WT; C194S and H673Q, DM) and then stimulated with mIFNβ (500 IU/ml) for 12 hrs. Data information: Data are representative of at least two biological replicates.


O Western blot analysis of Flag - Viperin in stable Flag - Viperin - expressing HEK293T cells transfected with shCtrl or shUSP33 and then treated with CHX ( 50 μM ) as indicated . Data information : Data are representative of at least two biological replicates .

O O 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 Western blot analysis of Flag-Viperin in stable Flag-Viperin-expressing HEK293T cells transfected with shCtrl or shUSP33 and then treated with CHX (50 μM) as indicated. Data information: Data are representative of at least two biological replicates.


D Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co - transfected with Flag - Viperin , HA - Ub and shUSP33 , then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological

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

D Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co-transfected with Flag-Viperin, HA-Ub and shUSP33, then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates


E Western blot analysis of Viperin levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV ( MOI = 1 . 0 , 12 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data are representative of at least two biological

O O O O O O O O O O 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

E Western blot analysis of Viperin levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates


G RT - qPCR analysis of VSV RNA levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data ( G ) represent mean and SD of three biological replicates , and p values were calculated using two - tailed unpaired Student ' s t - test For all statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) . * * * 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 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

G RT-qPCR analysis of VSV RNA levels in 2fTGH cells transfected with shCtrl or shUSP33 and then infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: Data (G) represent mean and SD of three biological replicates, and p values were calculated using two-tailed unpaired Student's t-test For all statistical testing: NS, not signiﬁcant (p > 0.05). ***p < 0.001.


I Survival curves of 8 - week - old Usp33 - / - mice fed an NSD ( 0 . 5 % NaCl ) or HSD ( 4 % NaCl ) for 7 days and then infected with VSV ( 1x108 PFU per gram body mouse , n = 10 ) . Data information : ; Data ( I ) show mean and SEM of ten biological replicates , and p value was calculated using logrank ( Mantel - Cox ) tests . For all statistical testing : NS , not signiﬁcant ( p > 0 . 05 ) . * * * p < 0 . 001 .

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I Survival curves of 8-week-old Usp33-/- mice fed an NSD (0.5% NaCl) or HSD (4% NaCl) for 7 days and then infected with VSV (1x108 PFU per gram body mouse, n=10). Data information: ; Data (I) show mean and SEM of ten biological replicates, and p value was calculated using logrank (Mantel-Cox) tests. For all statistical testing: NS, not signiﬁcant (p > 0.05). ***p < 0.001.


G Immunoprecipitation analysis of ubiquitination of Flag - USP33 in p97 - WT or p97 - KO 2fTGH cells co - transfected with Flag - USP33 and HA - Ub and then treated with NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

O O O O O 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 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

G Immunoprecipitation analysis of ubiquitination of Flag-USP33 in p97-WT or p97-KO 2fTGH cells co-transfected with Flag-USP33 and HA-Ub and then treated with NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


L Immunoprecipitation analysis of Viperin ubiquitination in HT1080 cells transfected with shCtrl or shp97 ( 1 # or 2 # ) and then treated with IFNα ( 1 , 000 IU / ml , 12 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : Data are representative of at least two biological replicates .

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

L Immunoprecipitation analysis of Viperin ubiquitination in HT1080 cells transfected with shCtrl or shp97 (1# or 2#) and then treated with IFNα (1,000 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates.


N Western blot analysis of Myc - Viperin in HEK293T cells co - transfected with Myc - Viperin and shCtrl or shp97 , and then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

O O O O 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

N Western blot analysis of Myc-Viperin in HEK293T cells co-transfected with Myc-Viperin and shCtrl or shp97, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


E , F Mass spectrometry analysis of acetylation of Flag - p97 ( E ) in RAW264 . 7 transfected with Flag - p97 and then treated with additional NaCl ( + 34 mM ) or control ddH2O ( Ctrl ) for 12 hrs . The intensity of p97 - K663 site was shown in ( F ) .

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E, F Mass spectrometry analysis of acetylation of Flag-p97 (E) in RAW264.7 transfected with Flag-p97 and then treated with additional NaCl (+34 mM) or control ddH2O (Ctrl) for 12 hrs. The intensity of p97-K663 site was shown in (F).


H Immunoprecipitation analysis of the interaction between USP33 and Myc - p97 in HEK293T cells transfected with Myc - p97 ( WT or K663R ) . Data information : Data are representative of at least two biological replicates .

O O O O O O O O O O O O O O O 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 Immunoprecipitation analysis of the interaction between USP33 and Myc-p97 in HEK293T cells transfected with Myc-p97 (WT or K663R). Data information: Data are representative of at least two biological replicates.


L Western blot analysis of FH - USP33 levels in p97 - KO HEK293T cells co - transfected with Myc - p97 ( WT or K663R ) and Flag - USP33 , and then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

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

L Western blot analysis of FH-USP33 levels in p97-KO HEK293T cells co-transfected with Myc-p97 (WT or K663R) and Flag-USP33, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


M Immunoprecipitation analysis of Viperin ubiquitination in p97 - KO 2fTGH cells co - transfected with Myc - p97 ( WT or K663R ) , Flag - Viperin and HA - Ub as indicated , and then treated with additional NaCl ( + 34 mM ) for 12 hrs . Data information : Data are representative of at least two biological replicates .

O O O O O O O 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 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

M Immunoprecipitation analysis of Viperin ubiquitination in p97-KO 2fTGH cells co-transfected with Myc-p97 (WT or K663R), Flag-Viperin and HA-Ub as indicated, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.


B Western blot analysis of USP33 in RAW264 . 7 cells cultured in media containing reduced concentration of NaCl ( - 17 and - 34 mM ) for 12 hrs . C Western blot analysis of Viperin in HT1080 cells treated with IFNα ( 1 , 000 IU / ml , 12 hrs ) in media containing reduced concentration of NaCl ( - 17 , - 34 and - 51 mM ) . Data information : Data are representative of at least two biological

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 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

B Western blot analysis of USP33 in RAW264.7 cells cultured in media containing reduced concentration of NaCl (-17 and -34 mM) for 12 hrs. C Western blot analysis of Viperin in HT1080 cells treated with IFNα (1,000 IU/ml, 12 hrs) in media containing reduced concentration of NaCl (-17, -34 and -51 mM). Data information: Data are representative of at least two biological replicates


D RAW264 . 7 cells were infected with VSV ( MOI = 1 . 0 , 12 hrs ) in media containing reduced concentration of NaCl ( - 17 and - 34 mM ) . Viral RNA levels were analyzed by RT - qPCR . Data information : Data ( D ) represent mean and SD of four biological replicates For all statistical testing : p values were calculated using two - tailed unpaired Student ' s t - test . N . S , not significant ( p > 0 . 05 ) and * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

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D RAW264.7 cells were infected with VSV (MOI=1.0, 12 hrs) in media containing reduced concentration of NaCl (-17 and -34 mM). Viral RNA levels were analyzed by RT-qPCR. Data information: Data (D) represent mean and SD of four biological replicates For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test).


H Mice ( n = 5 ) were intraperitoneally infected with VSV ( 1x108 PFU per gram body ) . 12 hrs post infection , mice were fed an NSD ( 0 . 45 % NaCl ) or LSD ( 0 . 15 % NaCl ) for 3 days . Then VSV RNA levels in mouse spleen tissues were detected by RT - qPCR . I Virus titers in the mouse blood ( n = 5 ) from ( H ) were analyzed by the TCID50 assay . Data information : Data show mean and SEM of five biological replicates . For all statistical testing : p values were calculated using two - tailed unpaired Student ' s t - test . N . S , not significant ( p > 0 . 05 ) and * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Mice (n=5) were intraperitoneally infected with VSV (1x108 PFU per gram body). 12 hrs post infection, mice were fed an NSD (0.45% NaCl) or LSD (0.15% NaCl) for 3 days. Then VSV RNA levels in mouse spleen tissues were detected by RT-qPCR. I Virus titers in the mouse blood (n=5) from (H) were analyzed by the TCID50 assay. Data information: Data show mean and SEM of five biological replicates. For all statistical testing: p values were calculated using two-tailed unpaired Student's t-test. N.S, not significant (p > 0.05) and **p < 0.01, ***p < 0.001 (two-tailed unpaired Student's t-test).


Associations between CSF T - tau , Ng and NFL ( panels A - C ) . Black squares : CN ( n = 109 ) , red circles : MCI ( n = 187 ) , blue triangles : AD ( n = 93 ) . Associations are shown for Spearman correlations . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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Associations between CSF T-tau, Ng and NFL (panels A-C). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Associations between CSF T - tau , Ng and NFL and between these biomarkers and CSF A42 ( panels D - F ) . Black squares : CN ( n = 109 ) , red circles : MCI ( n = 187 ) , blue triangles : AD ( n = 93 ) . Associations are shown for Spearman correlations . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Associations between CSF T-tau, Ng and NFL and between these biomarkers and CSF A42 (panels D-F). Black squares: CN (n=109), red circles: MCI (n=187), blue triangles: AD (n=93). Associations are shown for Spearman correlations. Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


CSF T - tau ( panel A ) , Ng ( panel B ) and NFL ( panel C ) in different combinations of clinical diagnosis and A pathology ( CN Aβ - , n = 69 ; CN Aβ + , n = 40 ; MCI Aβ - , n = 50 ; MCI Aβ + , n = 137 ; AD Aβ - , n = 8 ; AD Aβ + , n = 85 ) . See Table 5 for comparisons between groups . Biomarker levels are standardized to z - scores and shown in boxplots ( indicating median and interquartile ranges ; whiskers are defined as quartiles 1 and 3 + / - 1 . 5 * interquartile range , respectively ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

CSF T-tau (panel A), Ng (panel B) and NFL (panel C) in different combinations of clinical diagnosis and A pathology (CN Aβ-, n=69; CN Aβ+, n=40; MCI Aβ-, n=50; MCI Aβ+, n=137; AD Aβ-, n=8; AD Aβ+, n=85). See Table 5 for comparisons between groups. Biomarker levels are standardized to z-scores and shown in boxplots (indicating median and interquartile ranges; whiskers are defined as quartiles 1 and 3 +/- 1.5 * interquartile range, respectively). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).


Baseline associations between CSF T - tau , Ng and NFL and other AD traits . Data are estimates ( - coefficients ) from linear mixed effects models , with 95 % confidence intervals . The estimates are the main effects of the biomarkers , capturing the effects at study baseline . Effects were significant ( * ) for MMSE : T - tau ( p = . 034 ) and NFL ( p < . 0001 ) in A - negative . Note also that the range of the y - axes differs for the different outcomes , for visualization purposes . Models were adjusted for age and sex , and education ( for cognitive measures ) , and intracranial volume ( for MRI measures ) . Aβ42 and T - tau were measured using the the INNOBIA AlzBio3 kit ( Fujirebio , Ghent , Belgium ) , Ng was measured using an in - house immunoassay for Ng ( Portelius et al , 2015 ) and NFL was measured using the NF - light ® ELISA kit ( Uman Diagnostics , Umeå , Sweden ) .

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

Baseline associations between CSF T-tau, Ng and NFL and other AD traits. Data are estimates (-coefficients) from linear mixed effects models, with 95 % confidence intervals. The estimates are the main effects of the biomarkers, capturing the effects at study baseline. Effects were significant (*) for MMSE: T-tau (p=.034) and NFL (p<.0001) in A-negative. Note also that the range of the y-axes differs for the different outcomes, for visualization purposes. Models were adjusted for age and sex, and education (for cognitive measures), and intracranial volume (for MRI measures). Aβ42 and T-tau were measured using the the INNOBIA AlzBio3 kit (Fujirebio, Ghent, Belgium), Ng was measured using an in-house immunoassay for Ng (Portelius et al, 2015) and NFL was measured using the NF-light® ELISA kit (Uman Diagnostics, Umeå, Sweden).